Devices and assays for diagnosis of sinusitis

ABSTRACT

Methods and kits for sampling mucous from within a sinus to determine if a single sample includes one or more bacterial types indicating bacterial sinusitis.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 15/084,934, titled “DEVICES AND ASSAYS FOR DIAGNOSIS OFSINUSITIS,” AND FILED ON Mar. 30, 2016, which claims priority to U.S.Provisional Patent Application No. 62/140,405, titled “DEVICES ANDMETHODS FOR OBTAINING MUCOUS SAMPLES,” and filed on Mar. 30, 2015. Thispatent application also claims priority to U.S. Provisional PatentApplication No. 62/209,712, titled “DEVICES AND ASSAYS FOR DIAGNOSIS OFSINUSITIS,” and filed on Aug. 25, 2015. Each of these provisional patentapplications is herein incorporated by reference in its entirety.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

FIELD

The present application relates to methods and devices for thedetermination of the presence of one or more pathogens associated withbacterial sinusitis from a collected mucus sample, and preferably thedetection of three or more of the pathogens associated with over 90% ofbacterial sinusitis.

BACKGROUND

Sinusitis, defined as inflammation of the sinus tissues, usually as acomplication to viral infections from the common cold. Although thereare over 1 billion common colds in the U.S., a small percentage of themlead to sinusitis. In fact, 29 million people were diagnosed withsinusitis in 2011 in the US. Often antibiotics are ordered as atreatment for sinusitis and it is the 5th leading indication for theantibiotic prescriptions annually. Western EU markets are estimated tobe over 43 million patients annually. The majority of these patients areinitially seen by primary care physicians and then referred out tootolaryngologists, also known as ENT's if their symptoms do not resolve.Complicated cases of sinusitis eventually lead to surgery and there are1.5 million patients in the U.S. each year that are candidates forsurgical procedures, in which currently 500 k patients elect to undergosome type of surgical procedure. The direct costs association withmanaging sinusitis amount to over $6 billion annually, with another $3billion associated with indirect costs associated with sinusitismanagement.

The initial diagnosis of sinusitis remains a challenge for physicians. Apatient presenting at a physician's office with a symptom complex offever, headache and fatigue, also present in many different types ofsystemic diseases, could warrant a diagnosis of sinusitis. As a result,many patients with non-sinus related diseases such as migrainedisorders, chronic fatigue, and chronic systemic disorders aremisdiagnosed as sinusitis. An additional objective laboratory diagnostictesting would guide physicians as to the etiology of these commonsymptoms of viral upper respiratory tract infections, acute bacterialsinusitis and chronic sinusitis and lead to reduction of unnecessaryantibiotic and steroid prescriptions provided to patients.

Currently doctors typically decide on a treatment regime without adefinitive test to determine if the patient has viral sinusitis,bacterial sinusitis, upper respiratory infection, chronic fatigue, ormigraines, because it is difficult to diagnose the cause of sinusitis aseither viral or bacterial etiology. Treatment often involvesantibiotics, which are only effective for a small amount of theseconditions. The majority of sinusitis cases are viral, with someestimates that about 90% of sinusitis cases are viral. Majority of allpatients receive an antibiotic that they do not need, can make theircondition worse, and can lead to antibiotic resistance. Improved methodsof diagnosing sinusitis are needed. In particular, what is needed is adefinitive, rapid test for the cause of sinusitis, which could save thephysician time and provide timely information that will lead to fewerantibiotics being prescribed.

There are many advantages to determining the etiology of sinusitis(e.g., as viral, bacterial, etc.), including the reduction in healthcare costs, decreases in antibiotic use and concomitant bacterial drugresistance, and improvements in the level of care for patients.Described herein are bacterial sinusitis diagnostic apparatuses (e.g.,devices, systems, kits, etc.) and methods that may address many of theneeds described herein. For example, the sampling, testing, andtreatment apparatuses and methods described herein may allow for rapidand definitive diagnosis of bacterial sinusitis, permitting targetedtreatment with optimal antibiotics based on the specific diagnosis. Suchtargeted treatment may avoid unnecessary antibiotic treatments forpatients not suffering from bacterial sinusitis. A rapid diagnosis mayalso result in improved treatment for patients that test negative forbacterial sinusitis by instead treating the patient based on a negativetest for bacterial sinusitis.

SUMMARY OF THE DISCLOSURE

Described herein are apparatuses (e.g., systems, kits, assays, includinglateral flow assay kits) and methods which may allow determination ofthe presence of one or more of the three pathogens associated with over90% of bacterial sinusitis from a collected mucus sample. Specifically,these methods and apparatuses may determine, as part of a single rapidassay, the presence of one or more of: Haemophilus influenzae, Moraxellacatarrhalis and Streptococcus pneumoniae. In particular, describedherein are sinus collection devices for collection of mucus samples frompatient sinuses; these collection devices may be included as part of theassays described herein.

The sinus collection devices (sampling devices) described herein areintended for use during a routine office visit to a physician. Thesedevices may accurately and quickly (with a minimum of discomfort) allowthe acquisition of a mucus sample from the middle meatus region of thesinus (while avoiding miss-targeting of the region andcross-contamination). A collected mucus sample may then be analyzedusing any of the lateral flow assays described herein. If the test ispositive for any of the three bacterial pathogens, the patient hasbacterial sinusitis and may be prescribed an appropriate antibioticand/or steroid regimen to address the pathogenic bacteria. If the testis negative, the patient may be treated for viral sinusitis andantibiotics may not be administered. Examples of the sampling devicesand assays (e.g., lateral flow assays) are described herein. Althoughthe majority of these examples describe apparatuses, includingcollection devices, that are adapted for use in the nasal cavity, any ofthese apparatuses and methods may be adapted for use in other regions.For example, a variation of the sampling device and/or the assay may beadapted for use in collecting mucus samples from within the sinus duringsinus surgery procedures, from an ear (e.g., in the case of otitismedia, which is usually caused by the same three pathogens as isbacterial sinusitis) or elsewhere.

As will be described in greater detail below, these assays may beconfigured as lateral flow assays that include a single lysis solution(e.g., lysis buffer solution) that is appropriate for use with all threetypes of bacteria (e.g., H. influenzae, M. catarrhalis and S.pneumoniae) in order to expose the antigens specific to each one fordetection. Any of the assays described herein may be adapted for usewith the lysis buffer, and may include multiple (e.g., three) pairs, ordefined pools, of antigen binding agents that bind antigens (e.g.,surface proteins) specific to each type of bacteria (e.g., H. flu, M.cat, S. pneumo). The antigen binding agents (“agents”) may be monoclonalor polyclonal antibodies, or antibody fragments (e.g., FAB fragments,etc.) or molecules including all or a portion of these. Pairs of suchagents may bind to different portions of the same antigen. An agentspecific to each type of bacteria (e.g., H. flu, M. cat, S. pneumo) maybe bound to a solid phase substrate (e.g., membrane, particle, etc.) andspatially arranged in the assay and provide specific identification ofH. influenzae, M. catarrhalis and S. pneumoniae by visual detection ofbinding, including by binding the antigen to the tethered substrate andto a labeled agent. The pairs or pools of antibodies may be chosen tohave low cross-reactivity, while allowing comparable detection of H.influenzae, M. catarrhalis and S. pneumoniae.

The antigen binding agent (or “agent” and may also be referred to hereinas an indicator) may be chosen so that they are selective for theorganism of interest, binds cognate antigen specifically, have minimalcross-reactivity to common contaminating organisms and minimalcross-reactivity with commensal organisms. These antigen binding agentsmay also have a high affinity to the target pathogen antigen, rapidassociation kinetics, slow dissociation kinetics, and be sensitive tolow numbers of the pathogen. Finally these antigen binding agents may becompatible with lateral flow, and compatible with a conjugate. Asmentioned above, in particular the antigen binding agents may also becompatible for use with a common lysing solution for all threepathogens.

As will be described in greater detail herein, finding a common lysingsolution that may work with multiple types of pathogens, andparticularly H. flu, M. cat and S. pneumo, was surprisingly difficult,as many commonly used lytic agents (detergents, enzymes, etc.) did notwork with all three, resulting in incomplete lysis (clogging of thelateral flow system), lysis that was too slow (e.g., took longer than 15minutes), or disrupted the surface proteins, including the antigensspecific to each cell type.

Haemophilus influenzae (H. influenzae) may be detected using a pair orpool of antibodies that are specific to one or more antigen bindingagents that are relatively specific or characteristic of H. influenzae.For example, the indicator for H. influenzae may bind with specificityto the OMP-P2 and/or OMP-P5 antigen binding site for the pathogen. Asdescribed herein, numerous primary candidate antibodies have beenevaluated, and screened for cross reactivity between numerous (e.g., 30)commensal bacterial strains to assure minimal cross reactivity with thenormal flora occurring in the healthy sinus. Other examples of antigenbinding agents include antibodies that may be used are discussed inUS20140314876, herein incorporated by reference in its entirety.

Similarly, Moraxella catarrhalis (M. catarrhalis) may be detected usinga pair or pool of antigen binding agents that are specific to a markerfor M. catarrhalis (see, e.g., U.S. Pat. No. 7,811,589) such as ProteinC and Protein D outer member proteins.

One or more antigen binding agents specific for Streptococcus pneumoniae(S. pneumonia) may also be directed to S. pneumoniae markers such as thePsaA antigen.

Specifically described herein are assay kits for concurrently detectingH. influenzae, M. catarrhalis and S. pneumoniae from a mucosal samples.An assay kit may include: a lysis buffer to lyse cells within the sampleand form a single sample solution, wherein the lysis buffer comprisesbetween 0.01% and 5% (w/w) of the anionic surfactant and between 0.1%and 15% (w/w) of the osmotic agent; a cartridge containing one or moresolid phase substrates holding a first agent that that bindsspecifically to a first antigen specific to H. influenzae but not M.catarrhalis or S. pneumoniae, a second agent that binds specifically toa second antigen specific to M. catarrhalis but not H. influenzae or S.pneumoniae, and a third agent that binds specifically to a third antigenspecific to S. pneumoniae but not M. catarrhalis or H. influenzae,wherein the first, second and third agents are bound to specific regionsof the one or more solid phase substrates in the cartridge; one or moreconjugation regions within the cartridge, the one or more conjugationregions in fluid communication with the one or more solid phasesubstrates and comprising a fourth agent that is labeled and that bindsspecifically to the first antigen, a fifth agent that is labeled andthat binds specifically to the second antigen, and a sixth agent that islabeled and that bind specifically to the third antigen; one or moresample inlets on the cartridge in fluid communication with the one ormore conjugation regions; and one or more windows through which thespecific regions of the solid phase substrate to which the first, secondand third agents are bound may be visualized.

The anionic surfactant of the lysis buffer may comprise sarkosyl andwherein the osmotic agent of the lysis buffer comprises sucrose. Any ofthese assay kits may include a diluting buffer, as described herein.

The cartridge may include a housing that encloses one or more (e.g.,three, arranged in parallel) solid phase substrates. For example, acartridge may comprise a plurality (e.g., 3) of solid phase substrates,wherein each solid phase substrate holds one of the first agent, thesecond agent or the third agent. Alternatively, cartridge may comprise asingle solid phase substrate holding each of the first agent, secondagent and third agent. The first antigen may be a cell surface antigenspecific to H. influenzae, the second antigen may be a cell surfaceantigen specific to M. catarrhalis and the third antigen may be acell-surface antigen specific to S. pneumoniae.

Any of these cartridge regions may include a conjugation region. Theconjugation region may hold the unbound antigen binding agent, which maybe marked with a marker (e.g., a visualizable marker such as a colloidalmetal, colored bead, etc.). The antigen binding agent(s) in theconjugation region may be in solution (e.g., in a pre-wetted conjugationsponge or conjugation pad, a fluid conjugation chamber, etc.).Alternatively, the antigen binding agent (e.g., antibody, FAB, etc.) maybe lyophilized and stored in this region, and the sample solution mayre-suspend the antigen binding agent, allowing it to bind beforeentering the portion(s) of the solid phase substrate to to which antigenbinding agent(s) are bound. In variations having a single solid phasesubstrate with discrete regions for each of the different types ofantigen binding agents binding to specific bacterial types, a singleconjugation region (e.g., holding the fourth agent, fifth agent andsixth agent) may be used. Any of these cartridges may include multipleconjugation regions. In particular, cartridges having parallel fluidpaths may include multiple conjugation regions, where each conjugationregion holds the labeled antigen binding agent specific to one of thetypes of bacteria corresponding to the bound antigen binding agent onthe downstream solid phase substrate.

Any of these kits may include a cartridge a single sample inlet. Thesingle inlet may feed into a single fluidics line or into a plurality(e.g., 3) of parallel fluidic lines that may connect to, e.g., a sampleregion or chamber (e.g., sample pad), a conjugation region or chamber(e.g., conjugation pad), an incubation region or chamber (e.g.,incubation pad), a solid phase substrate region (e.g., detection region,which may be combined with the incubation region or chamber or separatefrom it), and/or a waste chamber or region (e.g., absorbent pad). Thefluid path(s) through the cartridge may include an air inlet. Forexample, an air inlet may be present at an opposite end of the fluidpath from the sample input.

The one or more windows in the cartridge may allow viewing of the solidphase substrate, allowing detection (e.g., visual, optical, etc.) ofbinding of antigen to the solid phase substrate(s) in this region (e.g.,the detection region) where the tethered/bound antigen binding agentspecifically bound to the solid phase substrate. In some variations themethod includes reading/detection of the binding using a readerincluding an optical reader (e.g., florescent reader, etc.), visual(e.g., manual or automatic) reading, etc. The cartridges describedherein may be configured to be compatible with one or more readers,including optical readers such as the Quidel “Sophia” device that is anoptical reader that uses fluorescent markers (see, e.g.,www.quidel.com/immunoassays/sofia-tests-kits) or the Becton Dickinson“Veritor” System (see, e.g.,www.bd.com/ds/veritorsystem/poctesting.asp).

As mentioned, any of the antigen binding agents (e.g., any or all of thefirst agent, second agent, third agent, fourth agent, fifth agent, andsixth agent) may comprise an antibody or an antibody fragment.

The one or more solid phase substrates may be, for example, a membraneor other surface onto which an antigen binding agent is immobilized. Thesubstrate may be smooth, porous, rough, etc. In some variations a singlesolid phase substrate is used to which each of the multiple antigenbinding agents (e.g., the first, second and third agents, each specificto an antigen of one of M. cat, S. pneumo, or H. flu). Thus, in any ofthese variations, the one or more conjugation regions may be a singleconjugation region, and the one or more sample inlets may be a singlesample inlet, and the single solid phase substrate may be upstream ofthe single conjugation region that is upstream of the single sampleinlet.

Any of these assay kits may also include a control region on the solidphase substrate. The control region may include an immobilized bindingagent that binds to one or more of the soluble antigen binding agents inthe assay (e.g., the first, second or third agent) configured to bind toone or more of the fourth agent, fifth agent, or sixth agent and anabsorbent pad, downstream of the specific regions of the solid phasesubstrate to which the first, second and third agents are bound.

For example, described herein are assay kits for concurrently detectingH. influenzae, M. catarrhalis and S. pneumoniae from a mucosal sample,the assay kit comprising: a lysis buffer to lyse cells within the sampleand form a single sample solution, wherein the lysis buffer comprisesbetween 0.01% and 5% (w/w) of the anionic surfactant and between 0.1%and 15% (w/w) of the osmotic agent; a cartridge containing a solid phasesubstrates holding a first agent that that binds specifically to a firstantigen specific to H. influenzae but not M. catarrhalis or S.pneumoniae, a second agent that binds specifically to a second antigenspecific to M. catarrhalis but not H. influenzae or S. pneumoniae, and athird agent that binds specifically to a third antigen specific to S.pneumoniae but not M. catarrhalis or H. influenzae, wherein the first,second and third agents are bound to specific regions of the solid phasesubstrate; and a conjugation region within the cartridge, conjugationregion in fluid communication with the solid phase substrate andcomprising a fourth agent that is labeled and that binds specifically tothe first antigen, a fifth agent that is labeled and that bindsspecifically to the second antigen, and a sixth agent that is labeledand that bind specifically to the third antigen; a sample inlet on thecartridge in fluid communication with the conjugation region; and one ormore windows exposing the specific regions of the solid phase substrateto which the first second and third agents are bound.

Also described herein are methods of concurrently detecting H.influenzae, M. catarrhalis and S. pneumoniae from a mucosal sample. Forexample a method of concurrently detecting H. influenzae, M. catarrhalisand S. pneumoniae from a mucosal sample may include: adding the sampleto a lysis buffer to lyse cells within the sample and form a singlesample solution, wherein the lysis buffer comprises both an anionicsurfactant and an osmotic agent; adding the sample solution to acartridge containing one or more solid phase substrates holding a firstagent that that binds specifically to a first antigen specific to H.influenzae but not M. catarrhalis or S. pneumoniae, a second agent thatbinds specifically to a second antigen specific to M. catarrhalis butnot H. influenzae or S. pneumoniae, and a third agent that bindsspecifically to a third antigen specific to S. pneumoniae but not M.catarrhalis or H. influenzae, wherein the first, second and third agentsare bound to specific regions of the one or more solid phase substratesin the cartridge; and contacting the sample solution, either before orafter it is added to the cartridge, with a fourth agent that is labeledand that binds specifically to the first antigen, a fifth agent that islabeled and that binds specifically to the second antigen, and a sixthagent that is labeled and that bind specifically to the third antigen.

In general, the agents that bind specifically to the antigens (e.g.,first antigen, second antigen, third antigen) described herein do notbind to antigens (proteins) from the majority of other commernsuralbacteria in the sinus specimen, in addition to having little or anybinding to other antigens other than the intended/target antigen. Forexample, the antigen binding agent (e.g., antibody or antibody fragment)may bind specifically to the target first antigen (e.g., from H. flu),but not to non-target antigens (e.g., from M. Cat or S. pneumo).

In any of these methods, kits and compositions described herein, thelysis buffer may comprise between 0.01% and 5% (w/w) of the anionicsurfactant and between 0.1% and 15% (w/w) of the osmotic agent. Theanionic surfactant of the lysis buffer may comprise between 0.01% and 5%(w/v) of sarkosyl and the osmotic agent of the lysis buffer maycomprises between 0.1% and 15% (w/w) of sucrose.

Any of these methods may include adding a diluting buffer to the samplesolution prior to adding it to the cartridge.

Adding the sample solution to the cartridge may include applying asingle bolus of sample or applying multiple boluses of sample. Forexample, adding sample solution to the cartridge may comprise dividingthe sample between a plurality of regions in the cartridge, wherein eachregion is in fluid communication with separate solid phase substratesand wherein each solid phase substrate holds one of the first agent, thesecond agent or the third agent.

Adding the sample solution to the cartridge may comprise adding thesample solution to a single region in the cartridge that is in fluidcommunication with a solid phase substrate holding each of the firstagent, second agent and third agent. In any of these methods, kits, andcompositions described herein, the antigens to each bacterial type maybe cell surface antigens. For example the first antigen may be a cellsurface antigen specific to H. influenzae, the second antigen may be acell surface antigen specific to M. catarrhalis and the third antigenmay be a cell-surface antigen specific to S. pneumoniae.

Any of these methods may include passing the sample solution over theone or more solid phase substrates in the cartridge after contacting thesample solution with the fourth, fifth and sixth agents.

The step of contacting the sample solution with the fourth, fifth andsixth agent may comprise passing the sample through one or more portionsof the cartridge upstream from the specific regions of the solid phasesubstrate in the cartridge to which the first, second and third agentsare bound.

Any of the methods described herein may include visually identifyingwhich strain (e.g., M. cat, S. pneumo, or H. flu) is present in thesample solution by identifying that the fourth agent has bound to thefirst antigen in the solid phase substrate region where the first agentwas bound, and/or the fifth agent has bound to the second antigen in thesolid phase substrate region where the second agent was bound, and/orthat the sixth agent has bound to the third antigen in the solid phasesubstrate region where the third agent was bound.

The sample solution may be exposed (e.g., contacted with) the labeledantigen binding agents either before or after it is added to thecartridge. For example, the sample solution may be contacted with thefourth agent, fifth agent, and sixth agent before it is added to thecartridge, or the sample solution may be contacted with the fourthagent, fifth agent, and sixth agent after it is added to the cartridge.

For example, a method for concurrently detecting H. influenzae, M.catarrhalis and S. pneumoniae from a mucosal sample may include: addingthe sample to a lysis buffer to lyse cells within the sample and form asingle sample solution, wherein the lysis buffer comprises between 0.01%and 5% (w/w) of the anionic surfactant and between 0.1% and 15% (w/w) ofthe osmotic agent; adding the sample solution to a cartridge containinga solid phase substrate holding a first agent that that bindsspecifically to a first antigen specific to H. influenzae but not M.catarrhalis or S. pneumoniae, a second agent that binds specifically toa second antigen specific to M. catarrhalis but not H. influenzae or S.pneumoniae, and a third agent that binds specifically to a third antigenspecific to S. pneumoniae but not M. catarrhalis or H. influenzae,wherein the first, second and third agents are bound to specificseparate regions of the solid phase substrate; contacting the samplesolution with a fourth agent that is labeled and that binds specificallyto the first antigen, a fifth agent that is labeled and that bindsspecifically to the second antigen, and a sixth agent that is labeledand that bind specifically to the third antigen; and visuallyidentifying through a window in the cartridge that the fourth agent hasbound to the first antigen, the fifth agent has bound to the secondantigen, or the sixth agent has bound to the third antigen.

Although the kits (e.g., assay kits, systems) described herein in theseexamples are configured to test for the presences of three bacteria(e.g., S. pneumoniae but not M. catarrhalis or H. influenzae), any ofthese kits and methods may be instead configured to identify thepresence of two or more than three bacteria. In particular, any of themethods and kits described herein may be configured to determine thepresence of S. pneumoniae and/or H. influenzae, which together accountfor approximately 70-75% of bacterial sinusitis.

Also described herein are nasal sampling devices that may be used bythemselves or as part of a kit or system for testing a nasal (e.g.,mucous) material, particularly from the middle meatus region of thesinus.

For example, a nasal sampling device for obtaining a sinus secretionsample from a subject's sinus may include: an elongate body having adistal end region that is bent relative to a proximal region by between15 degrees and 30 degrees; a sample collector on a distal end of anextendable shaft, wherein the sample collector is configured to collecta sample of sinus fluid, further wherein the sample collector is housedentirely within the distal end of the elongate body in a retractedposition; and a control coupled to the extendable shaft and configuredto extend and retract the sample collector in and out of the distal endof the elongate body; wherein the nasal sampling device has a retractedconfiguration with the sample collector retracted and housed entirelywithin the distal end of the elongate body, a sampling configurationwith the sample collector extended distally out of a distal opening ofthe distal end region of the elongate body a first distance between 0.5cm to 3 cm, and an elution configuration with the sample collectorextended distally out of the distal opening of the distal end region ofthe elongate body a second distance that is greater than the firstdistance.

A nasal sampling device for obtaining a sinus secretion sample from asubject's sinus, wherein the nasal sampling device includes: an elongatebody having a distal end region that is bent relative to a proximalregion by between 15 degrees and 30 degrees; a sample collector on adistal end of an extendable shaft, wherein the sample collector isconfigured to collect a sample of sinus fluid, further wherein thesample collector is housed entirely within the distal end of theelongate body in a retracted position; and a control coupled to theextendable shaft, the control having a first set point wherein thesample collector is extended distally out of a distal opening of thedistal end region of the elongate body a first distance between 0.5 cmto 3 cm, the control having a second set point, wherein the samplecollector is retracted and housed entirely within the distal end of theelongate body, the control having a third set point, wherein the samplecollector is extended distally out of the distal opening of the distalend region of the elongate body a second distance that is greater thanthe first distance.

Any of these nasal sampling devices may include a spacer (which may alsobe a protrusion, bump, deflector, etc.) on the extendable shaft proximalto the sample collector, wherein the spacer is configured to prevent thesample collector from contacting an inner surface of the elongate bodywhen the sample collector is retracted into the distal end of theelongate body. Centering the sample collector in this manner may preventthe sample collector from getting contaminated by other bacteria (e.g.,from regions other than the sampling region) by contacting the outerhousing of the elongate body, which may contact other regions; this mayalso prevent prematurely releasing material or limiting the amount ofmaterial held by the sample collector (e.g., swab).

Any of the nasal sampling devices described herein may include areleasable stop configured to prevent the control from selecting thethird set point until the stop is released. Any appropriate stop may beused, including an interference region between the extendable shaft andthe elongate body and/or handle, a latch, etc. For example, the stop maycomprise a detachable handle configured to releasably couple to a distalend of the extendable shaft. The stop may include a releasable connectorconnecting the extendable shaft to the stop.

In general, the dimensions of the nasal sampling may be configured foruse within the nasal passages (e.g., sinus) so that the sample collectormay be extended at the correct region of the apparatus to reach thedesired portion of the sinus (e.g. the middle meatus region, the uppermeatus region, the lower meatus region, etc.). Both the angle of thedistal end of the device relative to more proximal regions as well asthe size and shape of the device may be configured to allow external(through the nares/nostril) application of the device to sample themucosa. For example the distal end region of the elongate body may bebetween 1.5 and 3.5 cm long (e.g., between 1 and 5 cm long, between 1and 4 cm long, between 1.5 and 4 cm long, between 2 and 3 cm long,etc.). Similarly, the proximal region of the elongate body may begreater than 1 cm long (e.g., greater than 1.5 cm, greater than 2 cm,greater than 3 cm, greater than 4 cm, greater than 5 cm, between 1 cmand 30 cm, between 1 cm and 20 cm, between 1 cm and 15 cm, etc.).

Similarly, the sample collector may be any appropriate size (e.g.,between 0.2 and 2 cm long, between 0.4 and 1.5 cm long, between 0.5 and1.2 cm long, etc.). The extendable shaft may be any appropriate length(e.g., greater than 2 cm, greater than 5 cm, greater than 10 cm, between1 cm and 30 cm, between 1 cm and 20 cm, between 1 cm and 15 cm, between1 cm and 12 cm, etc.). The extendable shaft may be configured (byoperation of the control) to extend from the distal end region of theelongate body by a predetermined amount. For example, as mentionedabove, in a sampling position the sample collector may be extended fromthe distal end by between 0.5 cm to 3 cm. In the elution configurationthe extendable shaft is extended away from the elongate body furtherthan in the sampling configuration. This may be achieved by advancingthe extendable shaft relative to the elongate body, or by retracting thedistal end region of the elongate body proximally, relative to theextendable shaft, or in some variation by removing all or a portion ofthe distal end region of the elongate shaft. For example, in somevariations, the distance that the sample collector extends from theelongate body in the elution configuration (e.g., the second distance)may be 1.0 cm or greater than the first distance.

As described herein, in general the sample collector may be a swab,including in particular a flocked swab. It may also be beneficial to usea swab having ends which are branched (e.g., bifurcated, ormultiply-divided).

In any of these variations, the control on the nasal sampling device maybe coupled to a handle at the proximal end of the device. For example,any of these apparatuses may include a handle body extending proximallyfrom the elongate body, wherein the extendable shaft extends through theelongate body and into an internal channel within the handle body. Theextendable shaft may generally be a flexible elongate shaft. Theextendable shaft may be configured to slide within the elongate body.

Thus, any of the devices described herein may include a controlconfigured as a slider. Other examples of controls may include dials,knobs, switches, or the like. In some variations a control that may beincluded (e.g., in addition to a slider or other control) may be afinger ring. In some variations a control comprises may be a compressionactuator configured to be compressed to select the third set point inwhich the sample collector is extended distally out of the distalopening of the distal end region of the elongate body the seconddistance. In general, a control may be configured to be distallyadvanced to select the first set point in which the sample collector isextended distally out of a distal opening of the distal end region ofthe elongate body the first distance. In some variations a controlcomprises a push button configured to be depressed to select the thirdset point in which the sample collector is extended distally out of thedistal opening of the distal end region of the elongate body the seconddistance.

Any of these devices described herein may include a lock configured tolock the control at one or more of: the first set point, the second setpoint or the (optional) third set point.

Any of the devices described herein may include a depth gauge configuredto display a position of the sample collector to a user of the device.The distal end region may be configured to have an open configurationwhen the sample collector is advanced out of the distal end of theelongate body, and a closed configuration when the sample collector isin the retracted position.

Any of these devices may also include a depth stop to prevent thesampling device from being inserted too deep into a nasal and/or sinuscavity of a subject.

For example, a nasal sampling device for obtaining a sinus secretionsample from a subject's sinus may include: a hollow elongate body havinga distal end region that is bent relative to a proximal region bybetween 15 degrees and 30 degrees; a sample collector on a distal end ofan extendable shaft, wherein the sample collector is configured tocollect a sample of sinus fluid, further wherein the sample collector ishoused entirely within the distal end of the elongate body in aretracted position; a control coupled to the extendable shaft, thecontrol having a first set point wherein the sample collector isextended distally out of a distal opening of the distal end region ofthe elongate body a first distance between 0.5 cm to 3 cm, the controlhaving a second set point, wherein the sample collector is retracted andhoused entirely within the distal end of the elongate body, the controlhaving a third set point, wherein the sample collector is extendeddistally out of the distal opening of the distal end region of theelongate body a second distance that is 1.0 cm or greater than the firstdistance; and a projection on the extendable shaft proximal to thesample collector, wherein the projection is configured to prevent thesample collector from contacting an inner surface of the hollow elongatebody when the sample collector is retracted into the distal end of theelongate body.

Also described herein are methods including methods of using a nasalsampling device. For example, a method for detecting one or more nasalbacteria in a patient, using a nasal sampling device including anelongate body having a distal end region that is bent relative to aproximal region by between 15 degrees and 30 degrees, a sample collectoron a distal end of an extendable shaft, and a control coupled to theextendable shaft, the control having a first set point wherein thesample collector is extended distally out of a distal opening of thedistal end region of the elongate body a first distance, the controlhaving a second set point, wherein the sample collector is retracted andhoused entirely within the distal end of the elongate body, the controlhaving a third set point, wherein the sample collector is extendeddistally out of the distal opening of the distal end region of theelongate body a second distance that is greater than the first distance,may include: advancing the distal end region of the nasal samplingdevice through a nares of the patient until the distal end region isadjacent to a middle meatus of a sinus; setting the control to the firstset point to extend the sample collector into the middle meatus so thatit contacts a secretion fluid in the middle meatus; setting the controlto the second set point to retract the sample collector entirely withinthe distal end; withdrawing the nasal sampling device out of thepatient's nares; and testing the secretion fluid with an immunoassaytest after withdrawing the nasal sampling device.

The secretion fluid may be tested using any of the method describedabove (e.g., concurrently detecting H. influenzae, M. catarrhalis and S.pneumoniae from a mucosal sample). For example, testing the secretionfluid may include setting the control to the third set point, so thatthe sample collector is extended distally out of the distal opening ofthe distal end region of the elongate body a second distance that isgreater than the first distance and contacting the sample collector witha buffer solution. Testing the secretion fluid may comprise contactingthe secretion fluid with a lysing solution. For example, testing thesecretion fluid may comprise contacting the secretion fluid with alysing solution comprising both an osmotic agent and an anionicsurfactant. In some variations, testing the secretion fluid comprisescontacting the secretion fluid with a lysing solution comprising SodiumLauroyl Sarcosinate and sucrose to form a sample fluid and contactingthe immunoassay test with the sample fluid. Testing the secretion fluidmay comprise testing the secretion fluid with one or more agents thatbind to: an antigen specific to H. influenzae, an antigen specific to M.catarrhalis, or an antigen specific to S. pneumoniae. Testing thesecretion fluid may comprises testing the secretion fluid with one ormore agents that bind to each of: an antigen specific to H. influenzae,an antigen specific to M. catarrhalis, or an antigen specific to S.pneumoniae.

Also described herein are systems for detecting bacterial sinusitis thatgenerally include a mucosal sampling device as described herein any anyof the assays/kits described herein. For example, a system for detectingbacterial sinusitis may include a nasal sampling device for obtaining asinus secretion sample from a subject's sinus, wherein the nasalsampling device includes: an elongate body having a distal end regionthat is bent relative to a proximal region by between 15 degrees and 30degrees; a sample collector on a distal end of an extendable shaft,wherein the sample collector is configured to collect a sample of sinusfluid, further wherein the sample collector is housed entirely withinthe distal end of the elongate body in a retracted position; a controlcoupled to the extendable shaft, the control having a first set pointwherein the sample collector is extended distally out of a distalopening of the distal end region of the elongate body a first distance,the control having a second set point, wherein the sample collector isretracted and housed entirely within the distal end of the elongatebody, the control having a third set point, wherein the sample collectoris extended distally out of the distal opening of the distal end regionof the elongate body a second distance that is greater than the firstdistance; and an immunoassay kit for detecting at least one bacterialstrain associated with bacterial sinusitis infections.

The immunoassay kit may include a lysis buffer comprising both ananionic surfactant and an osmotic agent, such as an anionic surfactantbetween between 0.01% and 5% (w/w) and an osmotic agent between 0.1% and15% (w/w). In some variations the immunoassay kit may comprises a lysisbuffer comprising sarkosyl and sucrose.

In any of these variations, the immunoassay kit may include a cartridge,and the cartridge may include a sample inlet for depositing a sample, asample pad onto which the sample is absorbed prior to elution, aconjugate pad containing at least one antibody complexed with adetectable marker, a detector pad comprising at least one zone, whereinthe zone comprises antibodies directed to at least one bacterial antigenbound to the detector pad, and a visualization window for viewing theresults of the assay.

The immunoassay kit may comprise a cartridge comprising a sample inletfor depositing a sample, a sample pad onto which the sample is absorbedprior to elution, a conjugate pad containing a plurality of antibodiescomplexed with a detectable marker, a detector pad comprising aplurality of different zones, wherein each zone comprises antibodiesdirected to at least one bacterial antigen bound to the detector pad,and a visualization window for viewing one or more of the zones of thedetector pad. The kit may include a sampling device with a spacer on theextendable shaft proximal to the sample collector, wherein the spacer isconfigured to prevent the sample collector from contacting an innersurface of the elongate body when the sample collector is retracted intothe distal end of the elongate body.

A system for detecting bacterial sinusitis may include: a nasal samplingdevice for obtaining a sinus secretion sample from a subject's sinus,wherein the nasal sampling device includes: an elongate body having adistal end region that is bent relative to a proximal region by between15 degrees and 30 degrees; a sample collector on a distal end of anextendable shaft, wherein the sample collector is configured to collecta sample of sinus fluid, further wherein the sample collector is housedentirely within the distal end of the elongate body in a retractedposition; a control coupled to the extendable shaft, the control havinga first set point wherein the sample collector is extended distally outof a distal opening of the distal end region of the elongate body afirst distance, the control having a second set point, wherein thesample collector is retracted and housed entirely within the distal endof the elongate body, the control having a third set point, wherein thesample collector is extended distally out of the distal opening of thedistal end region of the elongate body a second distance that is greaterthan the first distance; and an immunoassay kit for detecting multiplebacterial strains associated with bacterial sinusitis infections, thekit comprising a lysis buffer comprising both an anionic surfactantbetween between 0.01% and 5% (w/w) and an osmotic agent between 0.1% and15% (w/w).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a healthy sinus and a sinus showingsymptoms of sinusitis.

FIG. 2 is a CT scan image of a patient exhibiting symptoms of sinusitis.

FIGS. 3A-3F show an example of a method for sampling a sinus inaccordance with some embodiments.

FIGS. 4A-4C illustrate aspects of a device configured to sample a sinusin accordance with some embodiments.

FIGS. 5A-5C illustrate aspects of a device configured to sample a sinusin accordance with some embodiments.

FIGS. 6A-6C illustrate aspects of a device configured to sample a sinusin accordance with some embodiments.

FIGS. 7A-7C illustrate aspects of a device configured to sample a sinusin accordance with some embodiments.

FIGS. 8A-8C illustrate aspects of a device configured to sample a sinusin accordance with some embodiments.

FIGS. 9A-9C illustrate aspects of a device configured to sample a sinusin accordance with some embodiments.

FIGS. 10A-10D illustrate aspects of a device configured to sample asinus in accordance with some embodiments.

FIGS. 11A-11C illustrate aspects of a device configured to sample asinus in accordance with some embodiments.

FIGS. 12A-12E illustrate aspects of a device configured to sample asinus in accordance with some embodiments.

FIGS. 13A-13E illustrate aspects of a device configured to sample asinus in accordance with some embodiments.

FIGS. 14A-14E illustrate aspects of a device configured to sample asinus in accordance with some embodiments.

FIG. 15A is a rendering of the lateral side of another variation of adevice configured to sample from a sinus.

FIG. 15B is a rendering of the posterior side of the assembled device ofFIG. 15A.

FIGS. 15C and 15D show partially exploded views of the lateral side andfront of the device of FIG. 15A.

FIG. 16 is an example of a proximal end of a sample collector beinginserted into a distal end of the main body portion.

FIG. 17A illustrates one variation of a distal end of a sample collectorand a corresponding sleeve into which the sample collector may behoused. Also shown is at least one coupler that joins the sleeve to themain body of the device.

FIG. 17B illustrates an alternative configuration for the at least onecoupler that joins the sleeve to the main body of the device.

FIG. 18A shows an exploded view of one variation of a distal end of ahandle and a proximal end of a main body region.

FIG. 18B illustrates a distal end of a thumb ring that is separated froma proximal end of a main body region of the device, similar to the viewof FIG. 18A. The thumb ring controller region at the distal end (leftside of FIGS. 18A and 18B) may be coupled into the distal end of themain body region.

FIG. 19 illustrates a sleeve for housing the distal end of the swab.Both the sleeve (protective cover) and the sample collector (includingswab) are bent in a predefined manner as described herein.

FIG. 20 shows a sample collector proximal end coupled with the distalend of the main body.

FIG. 21 illustrates a proximal end region of a sample collector thatcouples with the handle.

FIG. 22 illustrates another variation of a sampling device having acoupler and releasable hold (e.g. releasable lock, or release lock) onthe main body for engaging the distal handle.

FIG. 23A shows a coupler (shown as a snap-fit coupler) on the handle anda corresponding coupling channel on the main body.

FIG. 23B is an alternative view of the coupler of the handle and acorresponding coupling channel on the main body.

FIG. 23C is another view of a coupler of the handle and a correspondingcoupling channel on the main body.

FIGS. 24A-24D show schematics of one example of a lateral flow assay(having two detection readouts, e.g., for two sets of antibodies) andcomponents. FIG. 24A shows an assay housing, a sample pad for acceptingthe sample, a conjugate pad containing the first antibody with complexeddetector molecule, a detection pad along which the sample will run andcome into contact with zones of corresponding second antibodies bound tothe detection pad for each antigen of interest. FIG. 24B shows a sampleon the sample pad, the set of first antibodies on the conjugate pad, andzones on the detection pad holding different antibodies. FIG. 24C showsan eluting solution (dark) that runs across the detection pad and bringsfirst antibodies-detector molecule coupled to corresponding antigens incontact with the second set of antibodies. FIG. 24D shows the completedassay where the first antibodies-detector molecule coupled tocorresponding antigens is now also bound to the corresponding secondantibodies for each different antigen of interest.

FIGS. 25A-25K illustrate the operation of a sample collector asdescribed herein.

FIG. 26 schematically illustrates one variation of an assay similar tothe assay shown in FIGS. 24A-24D for diagnosing sinusitis.

FIG. 27 is a table illustrating the effectiveness of various lysisbuffers on three of the types of bacteria to be concurrently examined bythe apparatuses and methods described herein.

FIG. 28 is a table illustrating two examples of lysis buffers compatiblefor the concurrent detection of multiple different cell types (e.g., M.cat, S. pneumo and H. flu) as described herein.

FIG. 29 is a table illustrating two exemplary dilution bufferscompatible for the concurrent detection of multiple different cell typesas described herein. In these examples, the lysis buffer #1 (on left ofFIG. 28) was used with dilution buffer #1 (on left of FIG. 29), andlysis buffer #2 (on right in FIG. 28) was used with dilution buffer #2(on right in FIG. 29).

FIGS. 30A-30C illustrate detection of each of S. pneumo, M. cat, and H.flu, respectively, using the kits and methods described herein. Theconcentration of cells detected (expressed as colony forming units(CFU)/sample) in this prototype show thresholds for visual detectionfrom an exemplary lateral flow assay such as the one illustrated inFIGS. 24A-24D and 26. FIG. 30A illustrates that the prototype assaydetected the PsaA antigen (the cell-surface marker for S. pneumo) atbacterial concentrations ranging from 10³-10⁷ per 100 μl sample withresolution at 1×10⁴. FIG. 30B illustrates that the prototype assaydetected the CD antigen (a cell-surface marker for M. cat) at bacterialconcentrations ranging from 10⁴-10⁷ per 100 μl sample with goodresolution at 1×10⁵. FIG. 30C illustrates that the prototype assaydetected the OMP-P5 antigen (a cell-surface marker for H. flu) atbacterial concentrations ranging from 10⁵-10⁷ per 100 μl sample withgood resolution at 2×10⁵.

FIG. 31 is one example of a cartridge having a single solid phasesubstrate (combining three separate assays, one each for a differentbacterial type) that can simultaneously test for the presence of each ofthree different types of bacteria.

FIG. 32 is an example of a cartridge configured to simultaneously testfor the presence of each of three different types of bacteria inparallel; the cartridge include three separate solid phase substratesand three fluidic pathways. Although the example shown in FIG. 32includes three separate inlet ports, a single port having three fluidicpaths may be used.

DETAILED DESCRIPTION

Apparatuses (including devices, systems, kits, and assays) and methodsare disclosed herein for diagnosing sinusitis, including obtaining asample of sinus fluid from a patient and/or determining if the patientis infected with one or more of H. influenzae (H. flu), M. catarrhalis(M. cat) and S. pneumoniae (S. pneumo). For example, described hereinare sample devices for accurately and quickly sampling sinus fluidwithin the sinus, such as the middle meatus or maxillary sinus, andassays for rapidly testing this sample to determine the presence ofbacteria, viruses, and other diseases of interest. The fast diagnosis ofthe presence or absence of the diseases of interest can improve thetreatment of the patient.

FIG. 1 illustrates a comparison between a healthy sinus and a sinus withsinusitis. The sinusitis can cause excess mucous in the frontal sinusand maxillary sinus. Other symptoms can include inflamed sinus liningand a sinus infection. FIG. 2 illustrates a CT image of a patient withchronic sinusitis. The arrows indicate the congested sinuses typical ofchronic sinusitis.

Testing the mucous/sinus fluid within the sinus, such as the middlemeatus or maxillary sinus, can help diagnose the condition causing thediscomforting symptoms in the patient. The sinus fluid can indicate abacterial infection, viral infection, or provide other information tohelp diagnose and formulate an efficient and effective therapeutictreatment. Other examples of areas of the sinuses that can be testedusing the devices and methods disclosed herein are the frontal sinuses,maxillary sinuses, ethmoid sinuses, and sphenoid sinuses. The devicesdisclosed herein can also have a tip geometry configured to be advancedin other passages within the body. For example the devices can beconfigured to collect a sample from the nasopharynx region, esophagealpassage, from the middle ear, and other portions of the anatomy that askilled artisan would want to sample.

FIGS. 3A-3D show an example of a method for sampling a sinus inaccordance with some embodiments. FIGS. 3A-3D include a schematicillustrate of a portion of a sinus 100 including the nares 102, middlemeatus 103, ostium of the maxillary sinus 104, maxillary sinus 106, andsinus fluid 108 within the maxillary sinus 106. FIG. 3B is a schematicillustration of a portion of a sampling device 110. Any of the samplingdevices disclosed herein can be used as the sampling device 110 asillustrated in FIGS. 3A-3D. The sampling device 110 includes a distalportion configured to be advanced through the nares 102 to an areaadjacent to the middle meatus 103 and maxillary sinus 104 as shown inFIG. 3B. After the sampling device 110 has been advanced to a desiredarea adjacent to the middle meatus 103, the sample collector 112 can beadvanced distally to contact and sample sinus fluid in the middle meatus103 as shown in FIG. 3C. After the sample of the sinus fluid has beenobtained by the sample collector 112, the sample collector 112 can beretracted back into the sampling device 110. After the sample collector112 has been retracted back into the sampling device 110, the samplingdevice 110 can be withdrawn from the nares 102 as shown in FIG. 3D. Thesampling device 110 can be used to sample either of the nares.

After the sinus fluid has been sampled using the sampling device 110,the sinus fluid sample can be tested. FIG. 3E illustrates an example ofa kit that can include a sampling device as described herein. The kitcan include a lysis (e.g., buffer or lysis buffer) solution 150,diagnostic test 152, and packaging 154 in addition to the samplecollector. As will be described in greater detail below, a samplecollector 112 containing a sinus fluid sample can be advanced distallyas described herein, and placed in contact with the lysis buffersolution 150 to form the sample solution in which bacterial cells (andparticular the H. influenzae, M. catarrhalis and S. pneumoniae) will belysed to expose markers that can be detected by the assay. Thus, analiquot of the sample solution can be applied to the diagnostic test152. The diagnostic test 152 can produce a color change or otherindication visible to the medical technician to indicate a positive ornegative result for one or more of the bacteria tested (e.g., forsinusitis, H. influenzae, M. catarrhalis and S. pneumoniae). FIG. 3Fillustrates an example of a diagnostic test 156 with three differenttests (one each for H. influenzae, M. catarrhalis and S. pneumoniae) anda control. FIG. 3F illustrates an example of positive responses to allthree different tests and the control. In general, a diagnostic test 152can contain a plurality of immunoassay tests. The tests can providerapid results on the order of 1-30 minutes (e.g., 5-20 min, 5-17 min,5-15 min, etc.). Other configurations can be used for the immunoassaytests, for example multiple testing strips can be included in thediagnostic test 152 with each test strip testing for a differentpathogen on each strip, or some variation of a single sequential and oneor more parallel assays may be used. In some embodiments the diagnostictest includes tests for two or more pathogens. In some embodiments thediagnostic test includes tests for three or more pathogens. In someembodiments the diagnostic test includes tests for four or morepathogens.

In some embodiments the immunoassay tests can include common conditionsimplicated in sinusitis, such as strep A, influenza A, and influenza B.In some embodiments the immunoassay tests can include strep A. In someembodiments the immunoassay tests can include influenza A. In someembodiments the immunoassay tests can include influenza B.

In some embodiments the diagnostic tests can include bacterial sinusitistests. Examples of bacterial sinusitis pathogens include: Haemophilusinfluenzae, Moraxella catarrhalis, and Streptococcus pneumoniae. Otherexamples of diagnostic tests that can be used with the devices, kits,and methods disclosed herein include U.S. Patent Publication No.2014/0314876 to Das et al, titled “Proteomics Based Diagnostic DetectionMethod for Chronic Sinusitis”, the disclosure of which is incorporatedby reference herein in its entirety.

The sample collection devices disclosed herein can include a distal tipthat is configured to be advanced within the nare of the patient. Thedistal tip can include a bend that is configured to line up with theanatomy of most patients, such as the middle meatus. In some cases thebend has an angle of about 10 degrees to about 30 degrees relative to amajor axis of the device. In some embodiments the distal tip can beflexible. The distal tip can be made out of a soft, biocompatible, andpliable material, such as a polymer. In some embodiments the distal tipcan be made out of silicone. Other examples of biocompatible polymersinclude thermoplastic elastomer (TPE), thermoplastic vulcanizates (TPV),thermoplastic polyolefins (TPO), thermoplastic urethane (TPU) polymers,etc. Specific examples of polymers that can be used for the distal tipalso include Kraton, Versaflex, Santoprene, etc. Other biocompatiblepolymers know by the skilled artisan can also be used. In someembodiments the distal tip can be made out of metal. It may be desirable(though not necessary) to have a material hardness of between aboutDurometer Shore A90 to D85.

The distal tip can have an open end. In some embodiments the distal tipincludes a covered or closed distal end. The covered or closed distalend can be opened with distal advancement of the sample collector. Insome embodiments the covering can be designed to be punctured by thesample collector. In some embodiments the covering can be designed toopen and close to reduce the chance of contamination of the samplecollector. In some embodiments the covering or distal end can bedesigned to be resealably opened. For example, the cover or distal endcan have a patterned opening. The sample collector can be pushed throughthe patterned opening and the patterned opening can close after thesample collector is retracted. The closed distal end or covering canprevent contamination of the sample collector when the device isadvanced through the nare or retracted outside of the patient after thesample has been taken. In some embodiments the distal tip can have anopen distal end.

The sample collector can be advanced distally past a distal end of thedistal tip to take a sample of sinus fluid or other target fluid. Thesample collector can be a swab or contain another absorbent materialthat can collect and hold fluid. The advancement of the sample collectorcan be done using an actuator. In some embodiments the actuator can beslider or a plurality of sliders. In some embodiments a handle portionengaged with the sample collector can be used to advance and retract thesample collector. In some embodiments the mucous sample can be collectedusing negative pressure. For example, the actuator can create a negativepressure in the environment surrounding the distal tip such that themucous sample flows into the sample collector.

The device can include a safety or lock to reduce the inadvertentadvancement of the sample collector while the device is in the nare ofthe patient. For example, a button or slider can be required to bepressed to allow further advancement of the actuator. In someembodiments the slider itself can be required to be depressed before itcan slide. In some cases the safety can be a lock that can bedeactivated prior to further advancing the sample collector. In someembodiments the slider can include two sliders that are simultaneouslydepressed to allow movement of the actuator. In some cases the actuatorcan move along a track with notches to catch or stop the actuator at thesample position and sample solution position. In some cases the actuatorcan move along a track with a stair type configuration that requiresshifting the actuator at a stop position prior to further advancing orretracting the actuator.

In some embodiments the devices can be operated using a single hand. Forexample, one portion of the device can be held with one or more fingerswhile the actuator or proximal end of the device can be held andoperated using the thumb. The devices can be configured for ambidextroususe. For example, the device can be ergonomically designed toaccommodate use by the left hand and the right hand. The medicalprofessional can use whichever hand they prefer to operate the device.In some embodiments the device can be operated with both hands. Forexample, the lab technician may prefer to use both hands to extend thesample collector for processing.

The device can include a marker to indicate the orientation of thedevice, such as the direction of the bend in the distal end. The markercan indicate the lateral direction and/or the left or right nares. Themarker can include a colored portion of the device, a label on thedevice, or a projection on the exterior of the device indicating theorientation of the bend in the distal end.

The device can be used to take a sample from either nostril. Theorientation of the entire device can be rotated approximately 180degrees for use on the other nostril. In some embodiments the device canhave a rotatable portion that can be rotated, e.g. by 180 degrees, suchthat the device can be used for the other nostril. For example, thedistal portion of the tip can be rotated relative to the handle of thedevice.

The device can have a multi-piece construction. The sample collector canbe part of a removable handle. In some cases a portion of the handle canbe removed prior to being able to expose the sample collector to thesample solution. In some embodiments a portion of the distal cover canbe removed to access the sample collector.

The devices described herein can be used with an endoscope to provideadditional guidance and visualization to assist the healthcareprofessional with obtaining a sample from the desired location.

After obtaining the sample the device can be removed from the patientfollowed by contacting the sample collector with a sample solution. Thesample collector can be advanced distally past the distal end to contactthe sample collector with the sample solution. In some embodiments thesample collector can be withdrawn proximally through an interior of thedevice followed by contacting the sample collector with the samplesolution. In some embodiments the distal cover can be pulled back toexpose the sample collector. In some embodiments the distal cover canhave a multi-piece construction such that the cover can be removed toexpose the sample collector. In some embodiments a separate slider canbe used to advance the sample collector to a sample solution positionfor contact with the sample solution.

The device can include a depth gauge to provide information to the userregarding the location of the sample collector, such as the distance thesample collector has been advanced.

In some embodiments the device can include a stop or guard configured toengage with the outside of the nose/nostril to prevent furtheradvancement of the device. In some embodiments the stop or guard can beremoved by the healthcare worker to provide additional visual guidanceand clearance for endoscope

The devices can have a naturally retracted position. For example, acompression element could provide a resting force to keep the samplecollector in the retracted position. The compression element could pullthe sample collector proximally after obtaining the sample in theabsence of an actuating force applied by the user.

In some embodiments the hand held device can be configured to bedisposable after obtaining a sample fluid from the patient. In someembodiments the hand held devices can be configured to be reusable. Forexample, the device could be sterilized after obtaining a sample fluidand used for subsequent sample collection from a second patient. In someembodiments the handle can be designed to be reused and a new samplecollector or other part can be combined with the handle to form a devicefor obtaining a sample from a second patient. The sample collector couldbe provided separately as a single use cartridge to be used with thesterilized handle.

The sample collector can include a structure to facilitate openingand/or closing of a distal cover. For example, fins or a shoulder can belocated adjacent to the sample collector to push open the distal coverand to hold the distal cover open during retraction to prevent sampleloss caused by the distal cover squeezing the sample collector.

FIGS. 4-14 illustrate aspects of various embodiments of the hand heldsample collecting devices disclosed herein.

FIGS. 4A-4C illustrate aspects of a device configured to sample a sinusin accordance with some embodiments. FIGS. 4A-4C illustrate a hand heldsample collector 200 with a distal section 202 and a proximal section204. The distal section 202 includes a distal end 206 configured to beintroduced through the nares of the patient. The proximal section 204 isconfigured to slide relative to the distal section 202 to move thesample collector 210, illustrated as a swab, relative to the distal end206. The device 200 is configured to be gripped with a human hand with afinger grip 212 on the distal portion 202 and a thumb grip 214 on theproximal portion 204. The device 200 can be operated with one hand suchthat movement of a thumb on the thumb grip 214 can advance the proximalportion 204 relative to the distal portion 202. The device 200 includesan opening or window 216 such that a shaft 218 of the proximal portion204 can be observed. The window 216 can also be used to provideorientation information to the user, such as the lateral direction ofthe device. The illustrated device 200 includes a depth gauge 220 aidthe operator in determining the position of the sample collector 210.The distal portion 206 can be advanced through the nares to the targetlocation followed by advancing the proximal portion 204 and samplecollector 210 relative to the distal portion 206 to contact the sinusfluid. After the sample has been collected, the sample collector 210 isretracted back into the distal portion 206 of the device 200 to shieldthe sample collector 210 from the sinuses while withdrawing the device200. After the sample collector 210 has been retracted within the device200, the device 200 can be removed from the patient. The proximalportion 204 can be retracted proximally relative to the distal portion202 to completely separate the proximal portion 204 and the distalportion 202 for sample testing as shown in FIG. 4C. The proximal portion204 and sample collector 210 can be handled for the sample testing andprocessing. The sample collector 210 with the sinus sample can be testedusing the rapid diagnostic testing methods disclosed herein.

FIGS. 5A-5C illustrate aspects of a device configured to sample a sinusin accordance with some embodiments. FIGS. 5A-5C illustrate a hand heldsample collector 300 with a distal section 302 and a proximal section304. The distal section 302 includes a distal end 306 configured to beintroduced through the nares of the patient. A compression/springelement 308 engages with a shaft 310 connected to the sample collector312. The distal section 302 includes positioning markers 314. Thepositioning marker 314 can include a marker to provide an orientation ofthe device to the user, such as the “L” marking on the device 300indicating the lateral direction. A depth gauge 315 can be included onthe distal section 302 to provide depth positioning information to theuser. The device 300 can be gripped using the finger grip 316 andcompression element 308. The compression element 308 can be pushedforward to advance the shaft 310 and sample collector 312 distallyrelative to the distal portion 302 as shown in FIG. 5B to retrieve asample of sinus fluid. The compression element 308 provides a force toretract the sample collector 312 proximally in the absence of a forceapplied by the user. The compression element 308 can function as anautomatic retraction of the sample collector 312 after a sample of sinusfluid has been retrieved. The compression element 308 can be fullypushed forward to contact the sample collector 312 with the samplesolution as shown in FIG. 5C. Pushing the sample collector 312 distallyout of the device can minimize losses of the collected sinus fluid.

FIGS. 6A-6C illustrate aspects of a device configured to sample a sinusin accordance with some embodiments. FIGS. 6A-6C illustrate a hand heldsample collector 400 with a distal section 402 and a proximal section404. The distal section 402 includes a distal end 406 configured to beintroduced through the nares of the patient. The distal end 406 can beadvanced and retracted by an actuator, such as the slider 408. Theslider 408 can slide along the body of the proximal section 404. Theproximal section 404 includes a finger grip 410 and a thumb grip 411.The thumb grip 411 can pushed to advance the sample collector 412 asillustrated in FIG. 6B. The thumb grip 411 can retract in the absence ofan applied force to retract the sample collector back within the distalportion 406. The slider 408 can further retract the distal end 406 toexpose the sample collector 412 as shown in FIG. 6C for contact with asample solution while minimizing sample loss. The slider 408 can provideorientation of the distal end 406 to the user. For example, the distalend can be curved in the same direction/side as the location of theslider as shown in device 400. The bumps 414 on the distal portion 406can function as a depth gauge to provide additional positioning andorientation information to the user.

FIGS. 7A-7C illustrate aspects of a device configured to sample sinusfluid in accordance with some embodiments. The hand held samplecollector 500 includes a distal section 502 and a proximal section 504.The distal section 502 includes a distal end 506 with an outer covering508 having a patterned cut distal end section 510 that allows the samplecollector 512 to advance distally past the covering 508. The covering508 can be made out of a flexible and biocompatible material such assilicone. The device 500 includes a finger grip 514 and thumb grip 516.The thumb grip 516 can be advanced to push the sample collector 512distally past the covering 508 as shown in FIG. 7B. The sample collector512 advances past the patterned cut distal end section 510 to contactthe sinus fluid. The sample collector 512 can be retracted and coveredwhile removing the device from the user to prevent contaminating thesample collector with mucous from areas besides the targeted sinusfluid. The thumb grip 516 can include markings or a colored section 518to provide orientation information to the user, such as the direction ofthe bend in the distal section 502. The ridge 520 on the distal section502 can function as a depth gauge to provide additional positioning andorientation information to the user. The covering 508 can be pulled backto expose the sample collector 512 to the sample solution as shown inFIG. 7C. The covering 508 can be patterned or scored to fold back as itis pulled back away from the sample collector 512. Retracting thecovering 508 relative to the sample collector 512 can help minimize thesample loss from the sample collector 512.

FIGS. 8A-8C illustrate aspects of a device configured to sample a sinusin accordance with some embodiments. The hand held sample collector 600includes a distal section 602 and a proximal section 604. The distalsection has a two piece construction with a first distal sleeve portion606 and second distal sleeve portion 608. The proximal section 604includes a finger grip section 610 and a thumb grip 612. The finger gripsection 610 includes a marking 611 to label the lateral side of thedevice to let the user know the orientation of the bend in the distalsection 602 of the device 600. The thumb grip 612 can be pushed distallyto expose the sample collector 614 to collect a sample of sinus fluid asshown in FIG. 8B. The thumb grip 612 can be retracted to retract thesample collector 614 having the sinus fluid sample back within thedistal section 602 prior to removing the device 600 from the patient toavoid contaminating the collected sample. The sample collector 614 canbe processed after the device 600 is removed from the patient. Thesample collector 614 can be exposed to the sample solution by removingthe first distal sleeve portion 606 and second distal sleeve portion 608as shown in FIG. 8C.

FIGS. 9A-9C illustrate aspects of a device configured to sample a sinusin accordance with some embodiments. The hand held device 700 has a pentype shape with a distal section 702 and a proximal section 704. Thedistal section 702 includes a distal tip 706 with a patterned opening708. The sample collector 710 can be advanced past the patterned openingby advancing the slider 712. The device 700 includes two sliders 712 ona central portion 713 of the device. The body of the device 700 caninclude a marker 714 to provide information on the orientation of thedevice to the user, such as the direction of the bend of the distalsection 702. The device 700 can include a handle 716 on the proximalsection 704. The device 700 can be gripped by the user using the thumband middle finger. The slider 712 can have multiple positions. Aretracted position is shown in FIG. 9A. A partially advanced slider 712position is shown in FIG. 9B with the sample collector 710 advanceddistally past the patterned opening 708. The slider 712 can be advancedfurther to expose the sample collector 710 to the sample solution asshown in FIG. 9C. The slider 712 has a retracted position, sampleposition, and testing position. The device 700 can include a lock ateach of the retracted position, sample position, and testing position.The slider 712 can be configured such that both sliders 712 are pushedto allow movement of the slider 712. The device 700 can be used foreither nostril by rotating by 180 degrees. The distal tip 706 can bemade out of a soft polymer material like silicone to be comfortable forthe patient. The shape of the engagement 720 between the distal tip 702with a handle portion can be contoured as shown in FIGS. 9A-9C such thatthe contour of the engagement 720 can provide depth and orientationinformation to the user of the device. The patterned opening 708 canopen like an alligator jaw to protect the sample collector 710 frombeing contaminated by nasal mucous or bacterial or cross contaminationwhile the device is advanced in the nostril or retracted from thenostril after sampling. The device can include a shoulder or fin 718adjacent to the sample collector 710 to facilitate opening of patternedopening 708. The shoulder or fin 718 can push open the patterned opening708 and also assist with pushing open and holding open the patternedopening 708 when the sample collector 710 is retracted to minimizesample loss.

FIGS. 10A-10D illustrate aspects of a device configured to sample asinus in accordance with some embodiments. The hand held device 800 hasa distal section 802 and proximal section 804. The proximal section 804includes a handle 806 with a slider 812. The distal section 802 includesa distal portion 808. The distal portion 808 includes an orientationmarker 810 to display the orientation of the bend in the distal portion808. The distal portion 808 can be rotated 180° relative to the handle806 so that the device 800 can be used for either nostril. The distalportion 808 can be made out of a soft material such as silicone toimprove patient comfort. The slider 812 can be advanced to the samplemark 814 to distally advance the sample collector 816 past the distalportion 808 as shown in FIG. 10C. The sample collector 816 can befurther advanced using the slider 812 as shown in FIG. 10D to contactthe sample solution.

FIGS. 11A-11C illustrate aspects of a device configured to sample asinus in accordance with some embodiments. FIGS. 11A-11C illustrate ahand held sample collector 900 with a distal section 902 and a proximalsection 904. The distal section 902 includes a distal end 906 configuredto be introduced through the nares of the patient. The proximal section904 is configured to slide relative to the distal section 902 to movethe sample collector 910, illustrated as a swab, relative to the distalend 906. The device 900 is configured to be gripped with a human handwith a finger grip 912 on the distal portion 902 and a thumb grip 914 onthe proximal portion 904. The device 900 includes a marking 916 toindicate the orientation of the bend in the distal end 906. The devicecan include a collar 908 to prevent further advancement of the distalend 906 after it has been inserted in the nares. The device 900 can beoperated with one hand such that movement of a thumb on the thumb grip914 can advance the proximal portion 904 relative to the distal portion902. The distal end 906 can be advanced through the nares to the targetlocation followed by advancing the proximal portion 904 and samplecollector 910 relative to the distal portion 906 to contact the sinusfluid as shown in FIG. 11B. After the sample has been collected thedevice 900 can be removed from the patient and the proximal portion 904can be retracted proximally relative to the distal portion 902 tocompletely separate the proximal portion 904 and the distal portion 902as shown in FIG. 11C. The sample collector 910 with the sinus sample canbe tested using the rapid diagnostic testing methods disclosed herein.

FIGS. 12A-12E illustrate aspects of a device configured to sample asinus in accordance with some embodiments. The hand held device 1000 hasa pen type shape with a distal section 1002 and a proximal section 1004.The distal section 1002 includes a distal tip 1006 with a patternedopening 1008. The sample collector 1010 can be advanced past thepatterned opening by advancing the slider 1012. The device 1000 includestwo sliders 1012 on a central portion 1013 of the device. The body ofthe device 1000 can include a removable depth gauge 1018. The depthgauge 1018 can be rotated 90 degrees to a major axis of the device 1000to maximize visualization of the nasal cavity. FIG. 12B illustrates thedevice 1000 with the depth gauge 1018 removed. The body of the device1000 can include a marker 1014 to provide information on the orientationof the device to the user, such as the direction of the bend of thedistal section 1002. The device 1000 can include a handle 1016 on theproximal section 1004. The device 1000 can be gripped by the user usingthe thumb and middle finger.

The slider 1012 can have multiple positions. A retracted position isshown in FIGS. 12A and 12C. A partially advanced slider 1012 position isshown in FIG. 12D with the sample collector 1010 advanced distally pastthe patterned opening 1008. The slider 1012 can be advanced further toexpose the sample collector 1010 to the sample solution as shown in FIG.12E. Thus the slider 1012 has a retracted position, sample position, andtesting position. The device 1000 can include a lock at each of theretracted position, sample position, and testing position. The slider1002 can be configured such that both sliders 1012 are pushed to allowmovement of the slider 1012. The device 1000 can be used for eithernostril by rotating by 180 degrees. The distal tip 1006 can be made outof a soft polymer material like silicone to be comfortable for thepatient. The patterned opening 1008 can open like an alligator jaw toprotect the sample collector 1010 from being contaminated by nasal mucusor bacterial or cross contamination while the device is advanced in thenostril or retracted from the nostril after sampling. The device caninclude a depth gauge 1018 adjacent to the sample collector 1010. Thedevice 1000 can optionally include a safety that makes it difficult toaccidentally fully push the sample collector past the sampleconfiguration (FIG. 12D) while the device is deployed in the patient.The device 1000 can allow for improved endoscopic camera access for theuser. For example, the depth gauge 1018 can be removed to improveclearance for a camera or for visualization as shown in FIG. 10B.

FIGS. 13A-13E illustrate aspects of a device configured to sample asinus in accordance with some embodiments. The hand held device 1100 hasa distal section 1102 and a proximal section 1104. The distal section1102 includes a distal tip 1106 with a patterned opening 1108. Thesample collector 1110 can be advanced past the patterned opening byadvancing the handle 1113. The body of the device 1100 can include aremovable depth gauge 1118. FIG. 13B illustrates the device 1100 withthe depth gauge 1118 removed. The different appearance of the body ofthe device 1100 and the distal section 1102 can also provide depth andorientation information to the user. The body of the device 1100 caninclude a marker 1114 to provide information on the orientation of thedevice to the user, such as the direction of the bend of the distalsection 1102. The device can be held by the handle 1113 and finger grips1112. A retracted position is shown in FIGS. 13A and 13C. An advancedhandle 1113 position is shown in FIG. 13D with the sample collector 1110advanced distally past the patterned opening 1108. The handle 1113 canbe removed as shown in FIG. 13E. The bottom side of the device 1100includes a slider 1120 configured to advance the sample collector 1110to contact the sample solution as shown in FIG. 13E. The device 1100 canbe used for either nostril by rotating by 180 degrees. The device 1100can optionally include a safety that makes it difficult to accidentallyfully push the sample collector past the sample configuration while thedevice is deployed in the patient. The device 1100 can allow forimproved endoscopic camera access for the user.

FIGS. 14A-14E illustrate aspects of a device configured to sample asinus in accordance with some embodiments. The hand held device 1200 hasa distal section 1202 and a proximal section 1204. The distal section1202 includes a distal tip 1206 with a patterned opening 1208. Thesample collector 1210 can be advanced past the patterned opening 1208 byadvancing the handle 1213. The handle 1213 can be advanced and retractedby the user's thumb. The body of the device 1200 can include a removabledepth gauge 1218. FIG. 14B illustrates the device 1200 with the depthgauge 1218 removed. The different appearance of the body of the device1200 and the distal section 1202 can also provide depth and orientationinformation to the user. The body of the device 1200 can include amarker 1214 to provide information on the orientation of the device tothe user, such as the direction of the bend of the distal section 1202.The device can be held by the handle 1213 and finger grips 1212. Aretracted position is shown in FIGS. 14A and 14C. An advanced handle1213 position is shown in FIG. 14D with the sample collector 1210advanced distally past the patterned opening 1208. The handle 1213 canbe removed as shown in FIG. 14E. The bottom side of the device 1200includes a slider 1220 configured to advance the sample collector 1210to contact the sample solution as shown in FIG. 14E. In some embodimentsthe handle 1213 can be configured such that it has to be removed priorto being able to advance the slider. The device 1200 can be used foreither nostril by rotating by 180 degrees. The device 1200 canoptionally include a safety that makes it difficult to accidentallyfully push the sample collector past the sample configuration while thedevice is deployed in the patient. The device 1200 can allow forimproved endoscopic camera access for the user.

FIGS. 15A-21 illustrates devices configured to sample a sinus inaccordance with some embodiments. For example, in FIGS. 15A-15D, handheld device 1300 has a distal section 1302 and a proximal section 1304.Hand held device 1300 includes a sample collector 1310 housed within asleeve 1305 with a sleeve opening 1307, a main body 1330, a (thumb ring)handle 1313 and a depth stop 1318. Sleeve 1305 couples to main body 1330via a couplers, 1332. Couplers 1332 can have a double tip lock featureas shown in FIG. 17A or a single locking tip feature as shown in FIG.17B.

Sample collector 1310 (e.g., swab) can be advanced past sleeve opening1307 by the ring handle 1313, which may be configured to extend thesample collector a predetermined distance from the distal end. Thisfirst predetermined distance is configured to extend into the correctsample region (e.g., the sinus, such as the middle meatus or maxillarysinus), while avoiding regions distal to these regions which mayotherwise contaminate the sample. Handle ring 1313 includes a connector(shown as a handle ring cavity) 1315 that is able to engage a proximalend of the sample collector 1310 while both elements are retained withinmain body 1330. Sample collector includes at least one notch 1311 forcoupling to handle ring 1313 as shown in FIG. 16. FIG. 21 shows analternative design for coupling sample collector 1310 with handle 1313,which includes one or more notch 1311 (two are shown in this example)that is vertically arranged and parallel to the longitudinal axis ofsample collector 1310. Sample collector 1310 can be advanced past sleeveopening 1307 the first predetermined distance (e.g., between 5 and 20 mm(e.g., between 7 mm and 15 mm, between 8 mm and 12 mm, etc.) byadvancing handle 1313. Handle 1313 can be advanced and retracted by theuser's thumb. Having the handle 1313 limited to only advancing thesample collector 1310 the first predetermined distance from sleeveopening 1307 may prevent a longer portion of sample collector 1310 frombeing extended further into the sinus of the subject and causingdiscomfort and pain, and/or contamination. Once a sample has beencollected on sample collector 1310, sample collector 1310 may beretracted within sleeve 1305, and device 1300 removed from subject'ssinus and nasal cavity, the sample can be processed for assaying.

In both the designs shown in FIGS. 18A and 18B, handle 1313 can beremoved. Removal may also release a locking mechanism that prevents thesecond actuator/control (shown as slider 1312 in FIG. 15A) fromextending the sample collector. Removal may be achieved with minimalforce by pulling it in an opposite lateral direction from the body ofdevice 1300. Once handle 1313 has been removed from device 1300, slider1312, shown disposed longitudinally along device 1300 in FIG. 15A canadvance the sample collector 1310 beyond sleeve opening 1307 forprocessing a second predefined distance that is typically further thanthe first predefined distance. Thus, the distance that slider 1312 canadvance sample collector 1310 may be greater than the distance samplecollector 1310 can be extended with handle 1313. This secondpredetermined distance may allow the sample collector 1310 to beinserted into a sample (e.g., lysis buffer) tube for processing thesample, as described in greater detail below.

Any of these devices may also include a centering element 1340 (whichmay also be referred to a spacer or centering element) between thesample collector and the main body. Spacer 1340 in FIG. 17A is aprotrusion on either the sample collector (e.g., near the distal end) orthe main body that prevents sample collector 1310 from contacting theinner sides of sleeve 1305 when sample collector 1310 is extended andretracted from sleeve 1305. Preventing sample collector 1310 fromcontacting the internal sides of sleeve 1315 is especially importantwhen retracting sample collector 1310 because if the sample-containingsample collector 1310 scrapes past the internal sides of sleeve 1305 asit is being retracted, a portion of the sample collected on that surfaceof sample collector 1310 will be lost, thus diminishing the amount ofsample (e.g., cells or bacteria) available for processing and detection.

In the variations shown in FIG. 22, hand held sample device 1400 has adistal section 1402 and a proximal section 1404. Hand held device 1400includes a sample collector 1410 housed within a sleeve 1405 with asleeve opening 1407, a main body 1430, a (thumb ring) handle 1413 and adepth stop 1418.

Unlike the previously discussed embodiment where the handle is coupledto the sample collector with the main body, and where upon obtaining asample, the user can disengage the handle by pulling the handlelaterally away from the rest of the device, in the variations shown inFIG. 17, the device 1400 may include an additional safety feature toprevent inadvertent uncoupling of handle 1413 from sample collector 1410and main body 1430. In this variation, main body 1430 includes a channel1439. Channel 1439 is of a predetermined length with respect to mainbody 1430. In operation, handle 1413 couples with sample collector 1410and channel 1439 provides is of a predetermined length that handle 1413can only slide a given distance within main body 1430 when coupled. Thepredetermined distance corresponds to the distance sample collector 1410can extend from sleeve 1405. In any of the device variations describedherein, the handle may include a release 1442. Release 1442, whenengaged with channel 1439, may allow handle 1413 to extend and retractsample collector 1410 (e.g., the predetermined first length). To releasehandle 1413, a user presses and slide release element 1442 to allowrelease element 1442 to disengage from main body 1430 of device 1400.Then, similar to other embodiments, device 1400 includes a slider 1420that can extend sample collector 1410 a second predetermined distance(that may be greater than the first predetermined distance possible whensample collector 1410 is extended by handle 1413).

Methods of Using Extraction Device

Also disclosed herein are methods of using the devices described above,as well as sinusitis assays using them.

For example, the following steps can be taken to obtain a sample of apatient's infected mucous. When the patient is seated or laying down,place the sampling device (or other embodiment of the device) atapproximately forty five degrees with respect to the floor. Insertsleeve of the device (e.g., the distal end) into the subject's nasalpassage, ensuring that the angle of sleeve is pointed downward to followthe natural curvature of the nasal passage. Discretion should be usedwhere user experiences resistance when inserting the device into thenasal cavity of the subject. The depth stop on the device may provide asafety measure and prevent the user from inadvertently inserting more ofthe device into the subject's nasal cavity than is needed or safe.

Next, user pushes the slider forward to expose the swab's distal end tothe sinus middle meatus. In other embodiments, the thumb ring is coupledto the swab element. There, the user may use the thumb ring by insertinghis thumb through the thumb ring aperture and sliding the thumb ringforward to expose the distal end of the swab for sampling the sinusregion. Once a sample has been collected, the user can retract thesample collector distal end using the slider. In other embodiments, theuser can retract the distal end of the swab back into the sleeve bypulling the thumb ring proximal end away from the main body. The devicethen can be removed from the nasal cavity of the subject and the samplecollected can be now tested for bacterial presence.

FIGS. 25A-25K illustrate another method of operating the sinus mucosalsample devices described herein. In FIG. 25A, the sinus collectiondevice is inserted into the patient's nose by placing the device (probe)at an approximately 45 degree angle relative to the floor (shown byarrows), with the patient in an upright, sitting position. As shown inFIG. 25B, the collection device is then placed into the sinus byinserting the sinus collection device tip through the nasal cavity andinto sinus middle meatus until the depth stop of the device is engaged(e.g., the stop touches nasal entry), or until a slight tissueresistance is felt. FIG. 25C illustrates the collection device properlypositioned into the middle meatus region.

After positioning the collection device, as shown in FIG. 25D, the thumbring may be pushed to expose the swab tip and collect specimen, bypushing in the direction of the arrow to expose swab tip to sinusfluids. The tip of the swab is extended a first predetermined distanceinto the proper region of the sinus, as shown in FIG. 25E, showing theswab tip exposed for fluid collection. Thereafter, the swab tip may beretracted into collection device by reversing the thumb motion andretracting the swab tip into collection device, as shown in FIG. 25F.FIG. 25G shows the swab tip fully retracted into Collection device.Thereafter, the collection device may be removed from the nose, as shownin FIG. 25H. The collection device, once removed from nose, is ready forassay testing, which may be performed in a physician's office or otherlab area.

For processing, the distal controller, e.g., the distal handle or thumbloop, may be removed, as shown in FIG. 25i . Removing the handle in anyof these devices may release a limiter or lock that prevents the swabfrom extending distally. The collection device may be fully or partiallyextended before testing the sample. For example, FIG. 25K shows a devicewith the swab fully extended for processing; the swab portion (circled)was fully extended by sliding the second (distal) controller distally.Once fully extended, the swab may be processed by inserting into abuffer (e.g., lysing buffer) as described in greater detail below.

Sample Assay

Also described herein are assays that can be used to detect and diagnosebacterial sinusitis. More specifically, the assays may utilize antigenbinding agents (e.g., antibodies, antibody fragments, etc.) fordetecting markers specific to the types of bacteria contained with thesinus secretions of the subject. The methods disclosed herein may allowdetection of signature antigens that are associated with specificbacterial pathogens within the paranasal sinus cavity, and may thusallow a caregiver better insight as to whether prescribing an antibioticis beneficial. The assays may also provide information that aids acaregiver in deciding which antibiotic regimen would provide the mostfavorable outcome and most importantly, reduce the use of broad-spectrumantibodies in cases where such treatment would not effective.

These assays may utilize biomarkers that are specific antigensindicating the presence of the organism or pathological process ofinterest. “Biomarkers” are naturally occurring molecule, gene, orcharacteristic by which a particular pathological or physiologicalprocess, disease, or the like can be identified or characterized. Theterm “biomarker” may refer to a protein measured in sample whoseconcentration reflects the severity or presence of some disease state.Biomarkers may be measured to identify risk for, diagnosis of orprogression of a pathological or physiological process, disease or thelike. Exemplary biomarkers include proteins, hormones, prohormones,lipids, carbohydrates, DNA, RNA and combinations thereof. Although theexamples of assays described herein are specific to antigen bindingagents such as antibodies in a sandwich-type lateral flow assays, otherassays, including nucleotide hybridization, enzymatic andligand-receptor type assays may also or alternatively be used.

In some variations, the assay is capable of detecting at least onebiomarker, and more preferably two biomarkers, including biomarkers fromeach of a plurality of bacterial types linked to sinusitis. The assayscan be further modified to detect greater than two biomarkers (e.g.,preferably three). Furthermore, detecting the biomarkers can meandetecting a portion of the proteins, hormones, prohormones, lipids,carbohydrates, DNA, RNA and combinations thereof. The biomarkers mayalso be a biologically active variant of the naturally occurringmolecule of interest. For example, a protein or DNA biomarker can haveat least 65%, at least 70%, at least 80%, at least 85%, 86%, 87%, 88%,or 89%, and more typically 90%, 91%, 92%, 93%, 94%, and most common,95%, 96%, 97%, 98% or 99% conformity or sequence identity to the nativemolecule.

Assays and Kits

Any of the assays described herein may be part of a kit that allow auser to easily perform the assays for detecting antigens that are theprimary cause bacterial sinusitis. The kits may include the samplingdevice that is described above. The kits can also include a means forlysing the cells in order to expose the target antigens of interest,such as a lysis buffer, and a means for delivering the lysed supernatantto the assay portion of the kit.

A first critical step in obtaining accurate results is in properlyprocessing the sample extracted from the sampling device. Properprocessing includes formulating an appropriate lysis buffer. Whilefinding a lysis buffer that can lyse one particular type of cell isfairly straightforward, it is much more challenging to prepare a buffercomposition that is able to lyse multiple bacterial cells of interestwhile protecting the bioactivity of the antigens of interest and whilelysing in a reasonable amount of time (e.g., less than 15 minutes). Mostcells can be lysed by mechanical means, such as sonification orfreeze/thaw cycles, but such methods may require additional equipment.Thus, in some instances, it is preferable to use milder methods, such asdetergents, for disrupting the cell membrane. Detergents may disrupt thelipid layer surrounding cells by solubilizing proteins and interruptingthe lipid-lipid, protein-lipid, and/or protein-protein interactions. Theappropriate detergent composition also depends upon the type of cells tobe lysed, be it animal, bacteria, or yeast.

In the developing the “triple” assay for detecting one or more of M.catarrhalis, S. pneumoniae, and H. influenzae described herein, variouslysis buffers were tested for their ability to lyse all of the bacterialcells of interest, namely M. catarrhalis, S. pneumoniae, and H.influenzae. For example, while N-Lauroylsarcosine effectively lysed NTHIand exposed antigens for recognition by their antigens, it did noteffectively lyse M. catarrhalis. Next, TritonX-100, a commonly usedlysis buffer was also tested and appeared to lyse M. catarrhalis, butdid not work well with NTHI (nontypeable Haemophilus influenzae) Theinventors determined that the addition of sucrose to a lysis buffercontaining N-Lauroylsarcosine (e.g., Sarkosyl) was effective in lysingall three bacterial cell lines of interest. Without being limited to aparticular theory of operation, the addition of an appropriatepercentage of sucrose to the N-Lauroylsarcosine lysis buffer may providean osmotic shock to the cell membranes of the more lysis-resistant cellmembranes to achieve appropriate lysing.

FIG. 27 illustrates various examples of lysis buffers that have beenexamined, indicating their effectiveness with different types of thebacterial strains of interest. Although many of the buffers identifiedare effective in lysing one of the types of bacteria, in FIG. 27, onlythe 7% sucrose with 1.3% Sarkosyl was effective (and efficient) inlysing all three of M. catarrhalis, S. pneumoniae, and H. influenzae.For example, 0.1% Triton X-100 with lysozyme effectively/efficientlylysed M. catarrhalis but not S. pneumoniae or H. influenzae, while 1%Sarkosyl effectively/efficiently lysed H. influenzae but not S.pneumoniae, and M. catarrhalis. None of B-PER reagent, 0.1% Triton X-100without lysozyme, RIPA buffer, 0.1% Tween 20, 0.1% IGEPAL, 0.1% Tergitolor 0.1% Brij 35 was sufficiently effective in lysing any of M.catarrhalis, S. pneumoniae, and H. influenzae. Thus, it was surprisingthat only the combination of sucrose with Sodium lauroyl sarcosinate,(e.g., 7% sucrose with 1.3% Sarkosyl) was able to lyse all three typesof bacterial cells at near-comparable levels. Although only 7% sucrosewith 1.3% Sarkosyl, other combinations of sucrose (e.g., between 3% and15% sucrose, and between 0.5% and 3% Sarkosyl, etc.) may be effective.Thus, the combination of osmotic and anionic detergent disrupting cellwall appears to be most effective.

Thus, in one variation, a lysis buffer appropriate for use with all ofHaemophilus influenzae, Moraxella catarrhalis and Streptococcuspneumoniae includes between 5-15% sucrose (e.g., 7% sucrose), EDTA,PMSF, 1.3% sarkosyl (Sodium lauroyl sarcosinate), 50 mM Tris at a pH of8.0.

The assay may include antigen binding agents (e.g., antibodies, antibodyfragments, etc.) that specifically bind the protein biomarker ofinterest and components for immunoassay to detect the protein biomarkersusing associated antibodies. The kits can also contain instructions oncarrying out the sampling, performing the assay, and any of the methodsassociated with this invention.

The present invention provides for methods for detecting at least onebiomarker that is specific to a biofilm protein profile for a pathogenicbacteria. In general, immunological methods are well-known in the art,and performed routinely for diagnostic and research purposes. ELISA(enzyme-linked immunosorbent assay) is a powerful tool in studyingantibodies and antigens and their concentrations in a sample. ELISA canbe used to detect the presence of antigens that are recognized by anantibody or conversely, ELISA can be used to test for antibodies thatrecognize an antigen. An immunoassay that utilizing the ELISA platformis the two antibody sandwich ELISA.

Sandwich ELISA is used to determine antigen concentration in unknownsamples. If a pure antigen standard is available, the assay candetermine the absolute amount of antigen in an unknown sample. SandwichELISA requires two antibodies that bind to epitopes that do not overlapon the antigen. This can be accomplished with either two monoclonalantibodies that recognize discrete sites or a batch of affinity-purifiedpolyclonal antibodies. A purified first antibody (the capture antibody)is bound to a solid phase. A sample containing the corresponding firstantigen is added and allowed to complex with the bound first antibody.Unbound first antigen is washed away and a second antibody with a label(the detecting antibody) is allowed to bind to the first antigen, thusforming a “sandwich”. The assay is then either quantitative orqualitative amount of the second/detecting antibody bound. It is alsopossible to first bind the antigen to the labeled/detecting antibody andthen expose the antigen-labeled antibody complex to the bound antibody.

The present invention allows for greater than one sandwich ELISA assay,in order to concurrently detect one or more of Haemophilus influenzae,Moraxella catarrhalis and Streptococcus pneumoniae, which together mayaccount for >90% of bacterial sinusitis. In some variations, additionalpathogens may also be detected (e.g., Pseudomonas aeruginosa). In someembodiments, the assay contains two, three, or more distinct antigen andantibody pairings such that more than one antigen can be detected withone single assay. The results of the presence of at least one or moreantigen can be qualitatively obtained, meaning that there is a thresholdconcentration of the targeted antigen or antigens within the sample.Also, the assay can provide more quantitative measure of the presence ofone or more antigens by comparison with a standard or reference. Astandard or reference refers to a sample that has a known antigen andbiomarker, and in some cases, a known concentration of the known antigenand biomarker or antigens and corresponding biomarkers of interest.

An optical (including, but not limited to visual) indicator may be usedto indicate the presence of an antigen. The visual indicator istypically displayed on a region of the assay. The visual indicator canbe colorimetric. The visual indicator can also be a symbol, such as aline, that indicates the presence of a particular antigen. Theimmunoassay may contain labeling next to the regions where differentindicators for the presence of various antigens will be shown. Havingthe labeling will alert the user as to which particular antigen is orantigens are present with the sample. A caregiver user will then have abetter knowledge as to which antibiotic, if any, should be provided tothe subject.

The immunoassay can be of any suitable format. In some examples, theimmunoassay can be performed using a dipstick format where the samplesolution is drawn up the “dipstick” type assay with capillary action.The immunoassay can also have a largely horizontal format such as alateral flow assay. In this latter formats, the sample extracted fromthe subject's nose is treated such that the cells are lysed in anappropriate buffer, freeing the proteins and providing the samplesolution. An aliquot of the sample solution then can be placed in asample reservoir on the assay or other region noted on the assay andmigrated across regions of the assay that contain bound antibodies.Furthermore, the dipstick or flat format immunoassays can have a solidsupport made of any suitable material, such as nitrocellulose orpolyvinylidene difluoride (PVDF) or other membranes, dipstick, wells, ortubes.

In this present example, a combined immunoassay for three differentantigens are described. The three antigens of interest and describedbelow are all associated with bacterial sinusitis, but the overallconcept of having a multiple antigen test on one assay can be applied toother antigen/antibody systems as well. An example of a lateral flowassay 1500 is shown in FIGS. 24A-24D. Lateral flow assay 1500 includes ahousing 1509 having a sample inlet 1501 (e.g., sample port) fordepositing an aliquot of sample and detection window 1511 forvisualizing the results of the assay. A sample pad 1503 a conjugate pad1505, and a detection pad 1507 are included within a housing 1509.Sample pad 1503 will hold the sample to be run through the assay. Samplepad 1503 is in contact with conjugate pad 1505. Conjugate pad 1505contains the first antigen binding agent (e.g., shown here as anantibody) specific for at least one antigen. In some examples, theconjugate pad may contain two distinct antigen binding agent (e.g.,antibodies) that recognize and bind to two distinct antigens. In otherexamples, the conjugate pad may contain three distinct antigen bindingagents that recognize and bind three distinct antigens. When a sample isrun through sample pad 1503 past conjugate pad 1505, any antigen orantigens of interest will bind to corresponding antibodies. Theantibodies contained in conjugate pad 1505 may be linked to a detectormolecule. The complex or complexes of antigen with detector-labeledantigen binding agent is then delivered across detection pad 1507 wherea second antigen binding agent (e.g., antibody or antibodies)corresponding to the one more antigens are immobilized on a solidsupport. In this example, distinct antibodies are affixed in stripsacross detection pad 1507 as shown in FIGS. 24B and 24C. Thus, when thecorresponding antigen complexed with detector-labeled antibody is elutedpast the different regions of detection pad 1507, the antigen complexedwith detector-labeled antibody will bind to the corresponding secondimmobilized antibody and produce a signal as shown in FIGS. 24C and 24D.In any variation described herein, the pattern of the immobilizedantigen binding agent may be any appropriate pattern and/or density. Forexample, one (or preferably all) of the bound antigen binding agent maybe arranged on the solid phase substrate into a character, symbol,letter, word, pictogram, etc. In some variations the antigen bindingagent is arranged into a letter (e.g., spelling the initial or type ofbacteria (e.g., H. flu, M. cat, S. pneumo, etc.).

For example, an antigen profile for NTHI may include outer membraneproteins (OMP), specifically, OMP P5 and OMP P2. It has been verifiedthat presence of OMP P5 and OMP P2 within NTHI biofilm supernatant andthus detection of OMP P5 and OMP P2 with a sample is indicative of NTHIinfection. Corresponding antibodies were developed to both OMP P5 andOMP P2. For M. catarrhalis, antibodies to Protein C and Protein D outermember proteins (OMP-CD) may be used. For S. pneumoniae, the PsaA(pneumoccal surface adhesion A) protein may be a viable antigen toindicate the presence of S. pneumoniae. PsaA is a surface-exposed common37-kilodalton multi-functional lipoprotein detected on all knownserotypes of Streptococcus pneumoniae.

FIGS. 31 and 32 illustrate example of other types of cartridgeconfigurations that may be useful. For example FIG. 31 illustratesschematically how three different assays for different bacteria may becombined into a single assay, using a single lysis buffer for all threetypes of bacteria. In this example, the cartridge 3100 includes a singleport 3103. The cartridge also includes a window 3109 into an innerregion of the cartridge, showing the solid phase substrate on which theassay is being run.

FIG. 32 is another variation of cartridge in which each assay is run inparallel and the report out includes three separate windows and/or threeseparate solid phase substrates. Both of the examples shown in FIGS. 31and 32 include a control band that is formed as a positive control thatthe labeled antigen binding agent has diffused through the device. Asmentioned above, any of these device may include a vent or opening atthe end opposite to the cartridge.

Example 1 (Lysis Buffer)

As shown in FIG. 27, different lysis buffers were tested to determinethe best buffer composition for successfully lysing the cellscorresponding to Haemophilus influenzae (H. flu), Moraxella catarrhalis(M. cat) and Streptococcus pneumoniae (S. pneumo). Some of the potentialcandidates tested included N-Lauroylsarcosine, Triton X100, Sarkosyl,Sarkosyl and sucrose, and Bugbuster (Novogen). Standardized samples withknown concentrations of Haemophilus influenzae, Moraxella catarrhalisand Streptococcus pneumoniae were used to test the effectiveness of eachof these buffers. Based on the lysing data, the sucrose and Sarkosyllysis buffer composition appeared to be the most effective in being ableto lyse all three cell types. Importantly, many combinations (includingcombinations not including sarkosyl and sucrose) did not work for allthree cell types and therefore may not be compatible with a combinedassay for detection of all three cell types.

Thus, in general, only lysis buffers having an osmotic agent (e.g.,sucrose) and an anionic surfactant (sarkosyl, sodium lauroylsarcosinate, which may be referred to as an ionic surfactant, anionicdetergent or ionic detergent) was compatible with the assays for allthree of M. cat, S. pneumo, and H. flu. In particular, lysis buffershaving an osmotic agent between 0.1% and 15% (w/w or w/v, e.g., between0.5% and 12%, 0.5% and 10%, etc.) and an anionic surfactant between0.01% and 5% (w/w or w/v, e.g., 0.05% and 5%, 0.1% and 5%, 0.05% and 3%,etc.) were effective, whereas other lysis buffers having non-ionicdetergents/surfactants, enzymatic agents, or either osmotic agents aloneor anionic/ionic surfactants alone were not effective. For example thelysis buffer may include an osmotic agent within a range having a lowervalue of 0.1%, 0.2%, 0.3%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%,1.3%, 1.4%, 1.5%, 1.6%, 1.7%, and an upper value of 1.3%, 1.5%, 1.7%,2%, 2.5%, 3%, 4%, 5%, 7.5%, 10%, 12%, 15%, 20%, etc., where the lowervalue is less than the upper value, and an anionic detergent within arange having a lower value of 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%,0.35%, 0.4%, 0.45%, 0.5%, etc., and an upper value of 1.3%, 1.4%, 1.5%,1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 3%, 4%, 5%,7.5%, 10%, 12.5%, 15%, 17.5%, 20%, etc., where the lower value is lessthan the upper value. Examples of anionic surfactants (e.g., detergents)include alkylbenzenesulfonates, sulfates, sulfonates, and phosphateesters, including in particular sarkosyl (sodium lauroyl sarcosinate).Surprisingly, only lysis buffers containing the combination of anosmotic agent and an anionic surfactant within the specified ranges werecompatible for use in the assay looking for epitopes specific to each ofthe three cell types (M. cat, S. pneumo, H. flu).

FIG. 28 illustrates two exemplary lysis buffer that may be used (andwere used to generate exemplary readings in a lateral flow assays, suchas the ones shown in FIGS. 30A-30C, and may be used as part of the assay(e.g., kits, systems, etc.) described herein. FIG. 29 illustrates twoexemplary dilution buffers used as part of the assay (e.g., kits,systems, etc.) described herein.

Example 2 (Swabbing Material Selection)

Experiments testing the optimal sampling swab material was alsoperformed. Because the region where the sample is to be collected, asubject's nasal and sinus cavity, is a fairly sensitive area, it isimportant to be able to quickly and effectively gather enough samplematerial for assaying. Also, it would be desirable only sample thesubject's nasal and sinus cavities once because repeated sampling cancause irritation to the subject's nose and sinus cavities. In addition,any attempts to gather sample after a first try may elicit an autonomicresponse of excess mucous in the nasal passage that may dilute thesample collected or blood. While materials as cotton swabs and gauze canbe used, two commercially available swab materials were tested for theirability to quickly take up sample. Hydraflock, Ultraflock, and Purflockwere tested for their ability to take up water, ATS-M (artificial testsoil) lab soil containing mucin, and bacteria in ATS lab soil in a giventime interval. In addition to the uptake analysis, materials were alsoanalyzed for their ability to release sample. Hydraflock showed 100%recovery and release at 10 and 30 seconds. In contrast materials such asPurflock showed only 85% recovery and release.

Thus, in general, the sample collector (swab or swab material) may be aflocked material that is coupled to a shaft (e.g., an extendable and/orslideable shaft, rod, member, etc.). Flocked materials may minimizeentrapment of the cells, while efficiently absorbing them for laterrelease. In particular, flocked swabs having split (e.g., bifurcated, ormultiply-split) distal ends of the fibers, such as the hydroflockmaterial described above work surprisingly well, even compared to otherflocked materials.

Example 3: Assay

Following sampling with a device as described above for the collectiondevice, the swab with the sample is inserted into the lysis buffer byfully extending the swab tip and inserting into an appropriate volume oflysis buffer, as shown in FIG. 26. In this example, the swab positiveend goes into a tube with the lysis buffer (the swab may be the swab ofa sample device, such as shown in FIG. 25K). The swab may be mixed oragitated in the buffer, and removed (e.g., after between 1-30 sec). Thebacteria may be lysed within the sample buffer by the action of thelysing buffer. See FIGS. 28 and 29 for examples of lysing buffer (FIG.28) and dilution buffer (FIG. 29). The sample buffer may then bedirectly applied to the assay or it may be diluted (e.g., 1:10) in thelysis buffer or a second buffer (a dilution buffer, e.g., a Trisbuffer), which may aid in wicking on the membrane. 100-200 microlitersof sample may then be loaded into the port on a lateral flow cartridge,possibly followed by loading an additional volume (e.g., 100-200microliters) of buffer to induce wicking (as a “chaser”). An assayconfigured similar to that shown in FIGS. 24A-24D and described abovemay be used (e.g., a lateral flow assay) in which there are threeseparate regions arranged in sequence (adjacent each other) followed bya control region. This configuration is one variation of a multiplexedarrangement. Alternatively or additional one or more of the boundantibody-containing regions (sensing/detection regions) may be presentin a parallel region that is fed by the same or a different port. Inthese examples the antigen binding agents are antibodies, including apair of antibodies is directed to each antigen specific to one of thebacterial types; a tethered (e.g., in the detection region) antibody(e.g., antigen binding agent) and a soluble (detection) antibody(antigen binding agent) which may be linked to a visible marker(readout) such as colloidal gold or a visible dye (e.g., colored latexbead). In some variations the assay may be read in about 5 minutes(e.g., following 2-3 min of lysis or less and 3-5 min on the cartridgeor cassette). The time to a positive or negative result (as shown by thepositive control, which may be cross-reactive to the detection antibody,e.g., tethered anti-mouse antibody within a downstream control region),may depend on the wicking of the sample in the membrane. Typically afteradding the sample to the assay device (“cartridge” or “cassette”), thesample wicks through a sample pad into a conjugate pad, then into themembrane, where the markers may be captured by capture antibody,concentrating antibody also bound to the marker (antigen) and having areadout (e.g., colloidal gold) so that it can be visualized.

As mentioned, the capture antibodies may be laid down on the membrane indifferent characteristic positions (e.g., marked/labeled), and maydepend on the wicking capacity of the membrane.

FIGS. 30A-30C illustrate proof-of-concept data showing sensitivityranges for assays as described herein for detection of each of S.pneumo, M. cat, and H. flu, respectively.

In FIG. 30A, an assay using a first antigen binding agent that islabeled and that binds to OMP-P2 and/or OMP-P5 and a fourth tetheredantigen binding agent that binds specifically to another region of thesame antigen (e.g., OMP-P2 and/or OMP-P5) were used. The first andfourth antigen binding agents specific to H. influenzae but not M.catarrhalis or S. pneumoniae. In this example, a lateral-flow assay wasprepared with these antigen binding agents, a mucosal sample (which maybe taken using a sampling device such as the ones shown in FIGS. 15A-23Cand the steps of FIGS. 25A-25K). To generate the curve shown, the sampleused may be from a cultured example of the bacteria, so that theconcentration may be determined accurately. The sample was re-suspendeda lysis buffer such as the buffer shown in FIG. 28 (e.g., lysis Buffer#1), so that all three types of bacteria being sampled (e.g., H. flu, M.cat, and S. pneumo) were lysed appropriate following approximately 30sec to 1 min of lysis. The solution was then diluted and applied to thesample port 3201 of a lateral flow cartridge such as the one shown inFIG. 32, so that it may be fluidically channeled a conjugation region(e.g., chamber or bad) containing the labeled and untethered firstantigen binding agent. In this example, the antigen binding agent is amonoclonal antibody conjugated to a particle (e.g., colloidal gold ordyed bead) that can be visualized through the window of the cartridgeafter travelling (e.g., via capillary action) from the conjugationregion onto and/or across the portion of the solid phase structure towhich the fourth antigen binding agent is tethered. As illustrated inFIG. 30A, different concentrations of sample bacteria (H. flu) were usedto generate the sensitivity curve. In FIGS. 30A-30C, the detection wasperformed manually and visually, however higher sensitivity may beachieved by using a reader (e.g., optical reader) as mentioned above.

In this example, as described above, a single lysis buffer was used forlysing the sample so that multiple (e.g., M. cat, H. flu and S. pneumo)bacterial types could be simultaneously tested for from the same sample,even after a very brief lysis (e.g., between 5 seconds and 15 minutes,between 5 seconds and 10 minutes, between 5 seconds and 5 minutes,between 5 seconds and 4 minutes, between 5 seconds and 3 minutes,between 5 seconds and 2 minutes, between 5 seconds and 1 minute, between5 seconds and 45 seconds, etc., or less than 15 minutes, less than 10minutes, less than 5 minutes, less than 1 minute, etc.). The particularcomposition (and combination) of lysing agents described herein aresurprisingly effective at quickly, completely and gently lying themultiple different types/classes of bacteria without disrupting theantigens or their ability to be recognized by the antigen binding agentsused.

In FIG. 30B, M. cat was detected in parallel (e.g., by loading a sampleof the lysed solution into the second port 3207 of the cartridge shownin FIG. 32, and S. pneumo was detected by loading the solution into thethird port 3209 in FIG. 32. Alternatively concurrent detection may beperformed using a cartridge 3100 such as the one shown in FIG. 31. Withrespect to detection of Moraxella catarrhalis (M. cat) a pair of antigenbinding agents (second and fifth antigen binding agents or just agents)that are specific to the antigen Protein C (an outer member protein) atdifferent portion of the antigen were used; the second antigen bidingagent is labeled as described above (e.g., using colloidal gold), andthe fifth antigen binding agent is tethered to a specific region of thesolid phase substrate (e.g., a membrane within the cartridge). Similarlydetection of Streptococcus pneumoniae (S. pneumo) in FIG. 30C may beperformed using the lateral flow cartridge such as the one shown in FIG.32 (applying the sample into the port 3209). The antigen in this exampleis a PsaA antigen that is recognized by both the third and sixth antigenbinding agents, where the third agent is labeled and the sixth istethered. Thus, the second agent binds specifically to a second antigenspecific to M. catarrhalis but not H. influenzae or S. pneumoniae.Similarly, the third and sixth agents bind specifically to the thirdantigen specific to S. pneumoniae but not M. catarrhalis or H.influenzae.

In FIGS. 30A-30C, the sensitivities for detection of cells (expressed ascolony forming units (CFU)/sample) show thresholds for visual detectionbetween 10³-10⁵ per 100 μl sample. As mentioned above, this sensitivitymay be increased, for example by using a reader to read the cartridge.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.For example, as used herein, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. It will be further understood that the terms“comprises” and/or “comprising,” when used in this specification,specify the presence of stated features, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, steps, operations, elements, components, and/orgroups thereof. As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items and may beabbreviated as “/”.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if a device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

Although the terms “first” and “second” may be used herein to describevarious features/elements, these features/elements should not be limitedby these terms, unless the context indicates otherwise. These terms maybe used to distinguish one feature/element from another feature/element.Thus, a first feature/element discussed below could be termed a secondfeature/element, and similarly, a second feature/element discussed belowcould be termed a first feature/element without departing from theteachings of the present invention.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “about” or “approximately,” even if theterm does not expressly appear. The phrase “about” or “approximately”may be used when describing magnitude and/or position to indicate thatthe value and/or position described is within a reasonable expectedrange of values and/or positions. For example, a numeric value may havea value that is +/−0.1% of the stated value (or range of values), +/−1%of the stated value (or range of values), +/−2% of the stated value (orrange of values), +/−5% of the stated value (or range of values), +/−10%of the stated value (or range of values), etc. Any numerical rangerecited herein is intended to include all sub-ranges subsumed therein.

Although various illustrative embodiments are described above, any of anumber of changes may be made to various embodiments without departingfrom the scope of the invention as described by the claims. For example,the order in which various described method steps are performed mayoften be changed in alternative embodiments, and in other alternativeembodiments one or more method steps may be skipped altogether. Optionalfeatures of various device and system embodiments may be included insome embodiments and not in others. Therefore, the foregoing descriptionis provided primarily for exemplary purposes and should not beinterpreted to limit the scope of the invention as it is set forth inthe claims.

The examples and illustrations included herein show, by way ofillustration and not of limitation, specific embodiments in which thesubject matter may be practiced. As mentioned, other embodiments may beutilized and derived there from, such that structural and logicalsubstitutions and changes may be made without departing from the scopeof this disclosure. Such embodiments of the inventive subject matter maybe referred to herein individually or collectively by the term“invention” merely for convenience and without intending to voluntarilylimit the scope of this application to any single invention or inventiveconcept, if more than one is, in fact, disclosed. Thus, althoughspecific embodiments have been illustrated and described herein, anyarrangement calculated to achieve the same purpose may be substitutedfor the specific embodiments shown. This disclosure is intended to coverany and all adaptations or variations of various embodiments.Combinations of the above embodiments, and other embodiments notspecifically described herein, will be apparent to those of skill in theart upon reviewing the above description.

What is claimed is:
 1. A nasal sampling device for obtaining a sinussecretion sample from a subject's sinus, wherein the nasal samplingdevice includes: an elongate body having a distal end region that isbent relative to a proximal region by between 15 degrees and 30 degrees;a sample collector on a distal end of an extendable shaft, wherein thesample collector is configured to collect a sample of sinus fluid,further wherein the sample collector is housed entirely within thedistal end of the elongate body in a retracted position; and a controlcoupled to the extendable shaft, the control having a first set pointwherein the sample collector is extended distally out of a distalopening of the distal end region of the elongate body a first distancebetween 0.5 cm to 3 cm, the control having a second set point, whereinthe sample collector is retracted and housed entirely within the distalend of the elongate body, the control having a third set point, whereinthe sample collector is extended distally out of the distal opening ofthe distal end region of the elongate body a second distance that isgreater than the first distance.
 2. The nasal sampling device of claim1, further comprising a spacer on the extendable shaft proximal to thesample collector, wherein the spacer is configured to prevent the samplecollector from contacting an inner surface of the elongate body when thesample collector is retracted into the distal end of the elongate body.3. The nasal sampling device of claim 1, wherein the control comprises areleasable stop configured to prevent the control from selecting thethird set point until the stop is released.
 4. The nasal sampling deviceof claim 3, wherein the stop comprises a detachable handle configured toreleasably couple to a distal end of the extendable shaft.
 5. The nasalsampling device of claim 4, wherein the stop comprises a releasableconnector connecting the extendable shaft to the stop.
 6. The nasalsampling device of claim 1, wherein the second distance is 1.0 cm orgreater than the first distance.
 7. The nasal sampling device of claim1, wherein the sample collector comprises a swab.
 8. The nasal samplingdevice of claim 1, wherein the control is coupled to a handle at theproximal end of the device.
 9. The nasal sampling device of claim 1,further comprising a handle body extending proximally from the elongatebody, wherein the extendable shaft extends through the elongate body andinto an internal channel within the handle body.
 10. The nasal samplingdevice of claim 1, wherein the distal end region of the elongate body isbetween 1.5 and 3.5 cm long.
 11. The nasal sampling device of claim 1,wherein the proximal region of the elongate body is greater than 1 cmlong.
 12. The nasal sampling device of claim 1, wherein the extendableshaft comprises a flexible elongate shaft.
 13. The nasal sampling deviceof claim 1, wherein the extendable shaft is configured to slide withinthe elongate body.
 14. The nasal sampling device of claim 1, wherein thecontrol comprises a slider.
 15. The nasal sampling device of claim 1,wherein the control comprises a finger ring.
 16. The nasal samplingdevice of claim 1, wherein the control comprises a compression actuatorconfigured to be compressed to select the third set point in which thesample collector is extended distally out of the distal opening of thedistal end region of the elongate body the second distance.
 17. Thenasal sampling device of claim 1, wherein the control is configured tobe distally advanced to select the first set point in which the samplecollector is extended distally out of a distal opening of the distal endregion of the elongate body the first distance.
 18. The nasal samplingdevice of claim 1, wherein the control comprises a push buttonconfigured to be depressed to select the third set point in which thesample collector is extended distally out of the distal opening of thedistal end region of the elongate body the second distance.
 19. Thenasal sampling device of claim 1, further comprising a lock configuredto lock the control at one or more of: the first set point, the secondset point or the third set point.
 20. The nasal sampling device of claim1, further comprising a depth gauge configured to display a position ofthe sample collector to a user of the device.
 21. The nasal samplingdevice of claim 1, wherein the distal end region is configured to havean open configuration when the sample collector is advanced out of thedistal end of the elongate body, and a closed configuration when thesample collector is in the retracted position.
 22. The nasal samplingdevice of claim 1, further comprising a depth stop to prevent thesampling device from being inserted too deep into a nasal and/or sinuscavity of a subject
 23. A nasal sampling device for obtaining a sinussecretion sample from a subject's sinus, wherein the nasal samplingdevice includes: a hollow elongate body having a distal end region thatis bent relative to a proximal region by between 15 degrees and 30degrees; a sample collector on a distal end of an extendable shaft,wherein the sample collector is configured to collect a sample of sinusfluid, further wherein the sample collector is housed entirely withinthe distal end of the elongate body in a retracted position; a controlcoupled to the extendable shaft, the control having a first set pointwherein the sample collector is extended distally out of a distalopening of the distal end region of the elongate body a first distancebetween 0.5 cm to 3 cm, the control having a second set point, whereinthe sample collector is retracted and housed entirely within the distalend of the elongate body, the control having a third set point, whereinthe sample collector is extended distally out of the distal opening ofthe distal end region of the elongate body a second distance that is 1.0cm or greater than the first distance; and a projection on theextendable shaft proximal to the sample collector, wherein theprojection is configured to prevent the sample collector from contactingan inner surface of the hollow elongate body when the sample collectoris retracted into the distal end of the elongate body.
 24. A method fordetecting one or more nasal bacteria in a patient, using a nasalsampling device including an elongate body having a distal end regionthat is bent relative to a proximal region by between 15 degrees and 30degrees, a sample collector on a distal end of an extendable shaft, anda control coupled to the extendable shaft, the control having a firstset point wherein the sample collector is extended distally out of adistal opening of the distal end region of the elongate body a firstdistance, the control having a second set point, wherein the samplecollector is retracted and housed entirely within the distal end of theelongate body, the control having a third set point, wherein the samplecollector is extended distally out of the distal opening of the distalend region of the elongate body a second distance that is greater thanthe first distance, the method comprising: advancing the distal endregion of the nasal sampling device through a nares of the patient untilthe distal end region is adjacent to a middle meatus of a sinus; settingthe control to the first set point to extend the sample collector intothe middle meatus so that it contacts a secretion fluid in the middlemeatus; setting the control to the second set point to retract thesample collector entirely within the distal end; withdrawing the nasalsampling device out of the patient's nares; and testing the secretionfluid with an immunoassay test after withdrawing the nasal samplingdevice.
 25. The method of claim 24, wherein testing the sample comprisessetting the control to the third set point, so that the sample collectoris extended distally out of the distal opening of the distal end regionof the elongate body a second distance that is greater than the firstdistance and contacting the sample collector with a buffer solution. 26.The method of claim 24, wherein testing the secretion fluid comprisescontacting the secretion fluid with a lysing solution.
 27. The method ofclaim 24, wherein testing the secretion fluid comprises contacting thesecretion fluid with a lysing solution comprising both an osmotic agentand an anionic surfactant.
 28. The method of claim 24, wherein testingthe secretion fluid comprises contacting the secretion fluid with alysing solution comprising Sodium Lauroyl Sarcosinate and sucrose toform a sample fluid and contacting the immunoassay test with the samplefluid.
 29. The method of claim 24, wherein testing the secretion fluidcomprises testing the secretion fluid with one or more agents that bindto: an antigen specific to H. influenzae, an antigen specific to M.catarrhalis, or an antigen specific to S. pneumonia.
 30. The method ofclaim 24, wherein testing the secretion fluid comprises testing thesecretion fluid with one or more agents that bind to each of: an antigenspecific to H. influenzae, an antigen specific to M. catarrhalis, or anantigen specific to S. pneumonia.